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Effect of articaine on calcium transport in sarcoplasmic reticulum membranes isolated from medial pterygoid muscle.
Sánchez, Gabriel A; Di Croce, Daniel E; Richard, Susana B; Takara, Delia
Local anesthetics used in dentistry have myotoxic effects. Articaine, also known as carticaine, is one of the local anesthetics most widely used in clinical dentistry. The aim of this work was to describe its effect on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle. Ca-ATPase enzymatic activity was determined by a colorimetric method and ATP-dependent calcium uptake with a radioisotopic technique. Articaine inhibited both Ca-ATPase activity and calcium uptake in a concentration-dependent manner. Both inhibitory effects became evident at articaine concentrations lower than those employed in clinical dentistry. Half-maximal inhibitory concentrations (K) were 15.1 +/- 1.8 mM (n = 6) and 25.2 +/- 1.6 mM (n = 6) for enzymatic activity and calcium uptake, respectively. Preincubation of sarcoplasmic reticulum membranes with articaine enhanced Ca-ATPase activity in the absence of calcium ionophore, suggesting an ionophoric-like effect of the local anesthetic. We conclude that the inhibitory effect of articaine on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle is due to a direct interaction of the anesthetic with the enzyme and to the increased membrane permeability to calcium induced by this drug.
[ATPase activity and processes of calcium transport in membranes of sarcoplasmic reticulum of skeletal muscles with E-avitaminotic dystrophy].
Kurskiĭ, M D; Grigor'eva, V A; Medovar, E N; Meshkova, L I
Peculiarities of functioning of the sarcoplasmic reticulum muscles membranes with E-avitaminotic distrophy were studied. It was determined that the level of ATP-dependent consumption of Ca2+, value of the Mg2+, Ca2+-ATPase activity and an amount of the intermediate phosphorylated product forming in the reaction of ATP hydrolysis decrease. The rate of this product formation in the sarcoplasmic reticulum of the distrophic muscles is inhibited as compared to normalcy. Elimination of Ca2+ into calcium-free medium from the vesicular membranes of the reticulum preliminarily loaded with Ca2+ occurs more rapidly under dystrophy than in normalcy. The data obtained evidence for a disturbance of mechanism of Ca2+ active transport and for an increase in the membrane permeability for Ca2+ in the membranes of the dystrophic muscles sarcoplasmic reticulum. A problem is considered on a dependence of the skeletal muscles observed in the reticulum under dystrophy of the functional changes on the membrane structure, in particular on their lipid composition.
Observation of Network Dynamics of Ryanodine Receptors on Skeletal Muscle Sarcoplasmic Reticulum Membranes
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Hongli Hu
Full Text Available Rabbit muscle vesicles derived from sarcoplasmic reticulum were used as a material in studying networks of ryanodine receptors by cryo electron tomography. Three-dimensional analysis reveals the dynamical features of these networks. It was found that the connection angles were rotated along the transmembrane axis of ryanodine receptors. Majority of the connections was observed at domains 6/6 of ryanodine receptors while a small group of connections were showed at domains 9/10. The flexible rotation and connection shift seem to facilitate the extension of an annular network on the wall of the sarcoplasmic reticulum in a triad.
The interactions between mitochondria and sarcoplasmic reticulum and the proteome characterization of mitochondrion-associated membrane from rabbit skeletal muscle.
Liu, Zhouying; Du, Xiangning; Deng, Jie; Gu, Mingyue; Hu, Hongli; Gui, Miao; Yin, Chang-Cheng; Chang, Zhenzhan
To obtain a comprehensive understanding of proteins involved in mitochondrion-sarcoplasmic reticulum (SR) linking, a catalog of proteins from mitochondrion-associated membrane (MAM) of New Zealand white rabbit skeletal muscle were analyzed by an optimized shotgun proteomic method. The membrane fractions were prepared by differential centrifugation and separated by 1D electrophoresis followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ)-Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 459 proteins were identified from both of the two independent MAM preparations. Protein pI value, molecular weight range, and transmembrane region were calculated using bioinformatics softwares. One hundred one proteins were recognized as membrane proteins. This protein database suggested that the MAM preparations composed of proteins from mitochondrion, SR, and transverse-tubule. This result indicated mitochondria physically linked with SR in rabbit skeletal muscle, voltage-dependent anion channel 1 (VDAC1), VDAC2, and VDAC3 might participate in formation of the tethers between SR and mitochondria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles.
Riley, R T; Goeger, D E; Yoo, H; Showker, J L
Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.
Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle.
Bowman, B J; Berenski, C J; Jung, C Y
Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.
Altered calcium pump and secondary deficiency of γ-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from δ-sarcoglycan knockout mice
Solares-Pérez, Alhondra; Álvarez, Rocío; Crosbie, Rachelle H.; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J.; Ortega, Alicia; Coral-Vazquez, Ramón
Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and δ-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of δ-SG isoforms in TT and SR results in a secondary deficiency of γ-SG and µSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the δ-SG isoforms may stabilize the expression of γ-SG and µSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. PMID:20638123
The inhibitory influence of calix[4]arene of C-90 on the activity of Ca(2+,Mg(2+-ATPases in plasma membrane and sarcoplasmic reticulum in myometrium сells
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T. O. Veklich
Full Text Available Our study on the plasma membrane vesicles and myometrium cells treated with 0.1% digitonin showed that inhibitory effect of calix[4]arene C-90 (5,11,17,23-tetra(trifluoromethyl(phenylsulphonylimino-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene on the plasma membrane Ca2+,Mg2+-ATPase was more significant than on the Ca2+,Mg2+-ATPase in sarcoplasmic reticulum (the inhibition coefficient I0.5 values were 20.2 ± 0.5 µM and 57.0 ± 1.4 µM for the plasma membrane Ca2+,Mg2+-ATPase and Ca2+,Mg2+-ATPase in sarcoplasmic reticulum, respectively (n = 5. Inhibition kinetics of calix[4]arene C-90 effect on the Ca2+,Mg2+-ATPase activities in plasma membrane and sarcoplasmic reticulum were studied. This substance inhibited both pumps as complete noncompetitive inhibitor. Calix[4]arene C-90 caused the increase of intracellular Ca2+ concentration and decrease of hydrodynamic diameter in smooth muscle cells similar to the action of uterotonic drug oxytocin.
Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles
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Campbell, Kevin Peter [Univ. of Rochester, NY (United States)
Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).
Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters
Nicholl, Peter A.; Howlett, Susan E.
Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes.…
Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.
Hymel, L; Maurer, A; Berenski, C; Jung, C Y; Fleischer, S
The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.
Inhibitory effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent atpase from temporalis muscle.
Sánchez, Gabriel A; Casadoumecq, Ana C; Alonso, Guillermo L; Takara, Delia
Myotoxic effects of local anesthetics on skeletal musclefibers involve the inhibition ofsarcoplasmic reticulum Ca2+ -dependent ATPase activity and Ca2 transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+ -dependent ATPase isolated from rabbit temporalis muscle. Ca2+ -dependent ATPase activity was determined by a colorimetric method Calcium-binding to the Ca dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+ -dependent ATPase activity in a concentration-dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+ dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+ -binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+ -dependent ATPase from temporalis muscle is due to the drug's direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.
Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles
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Campbell, K.P.
Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.
Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle
DEFF Research Database (Denmark)
Frías, J A; Cadefau, J A; Prats, C
damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may......Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit...... muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose...
Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy
Paran, Christopher W.; Zou, Kai; Ferrara, Patrick J.; Song, Haowei; Turk, John; Funai, Katsuhiko
Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) that contributes to abnormal Ca2+ homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1 and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition. PMID:26361872
Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.
Paran, Christopher W; Zou, Kai; Ferrara, Patrick J; Song, Haowei; Turk, John; Funai, Katsuhiko
Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition. Copyright © 2015. Published by Elsevier B.V.
Sub-sarcolemmal swelling of sarcoplasmic reticulum after isometric contractions in rat semimembranosus lateralis muscle
Willems, M.E.T.; Huijing, P.A.J.B.M.; Friden, J.
The decline in isometric force, swelling of sarcoplasmic reticulum and loss of desmin was measured in semimembranosus lateralis muscle of male Wistar rats immediately after a short series of brief (500 ms) maximal isometric contractions. For the active muscle, the series ended below (protocol A) and
Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle.
Sánchez, Gabriel Antonio; Di Croce, Daniel Eduardo; Casadoumecq, Ana Clara; Richard, Susana Beatriz; Takara, Delia
The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by P(i) were determined with radioisotopic techniques. Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5 μM Ca(2+), 100 μM EGTA, 90 μM Mg(2+), 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by P(i) was lower. The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles. Copyright © 2012 Elsevier Ltd. All rights reserved.
Time course of activation of calcium release from sarcoplasmic reticulum in skeletal muscle.
Simon, B J; Schneider, M F
Myoplasmic free calcium transients were measured with antipyrylazo III in voltage clamped segments of frog skeletal muscle fibers and were used to calculate the rate of release (Rrel) of calcium from the sarcoplasmic reticulum. Intramembrane charge movement was measured for the same pulses in the same fibers. During a depolarizing pulse Rrel rose to an early peak and then decayed relatively rapidly but incompletely due to calcium-dependent inactivation (Schneider M.F., and B.J. Simon. 1988. J...
Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures.
Charuk, J H; Guerin, C.; Holland, P.C.
We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumu...
Effects of high pressure treatment on Ca2+ release and Ca2+ uptake of sarcoplasmic reticulum.
Okamoto, A; Suzuki, A; Ikeuchi, Y; Saito, M
To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca2+ release from SR in the rabbit skeletal muscle treated with high pressure (100-300 MPa, 5 min) were investigated in comparison with those of the SR from conditioned muscle. The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS-PAGE profile were not observed in the SR from the pressurized muscle up to 200 MPa, but a marked decrease of the ATPase protein and high-affinity Ca(2+)-binding protein were observed in the SR from the pressurized muscle at 300 MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200 MPa. When the muscle was pressurized at 300 MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle. Ca2+ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca2+, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle. It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)
Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.
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Yael Yaniv
Full Text Available Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP.To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84.A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.
Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle.
Misquitta, C M; A Sing; Grover, A. K.
Cardiac muscle expresses sarcoplasmic/endoplasmic-reticulum Ca2+ pump isoform SERCA2a; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100-200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (mean+/-S.E.M.) were (29+/-4)-fold higher in the heart....
Alterations in mitochondria and sarcoplasmic reticulum from heart and skeletal muscle of horizontally casted primates
Sordahl, L. A.; Stone, H. L.
Horizontally body-casted rhesus monkeys are used as an animal model in order to study the physiological changes known as cardiovascular deconditioning which occur during weightless conditions. No difference was found between the experimental and control animals in heart mitochondrial oxidative phosphorylation which indicates that no apparent changes occurred in the primary energy-producing system of the heart. A marked increase in cytochrome oxidase activity was observed in the casted primate heart mitochondria compared to controls, while a 25% decrease in respiratory substrate-supported calcium uptake was found in casted primate heart mitochondria compared to controls. Sacroplasmic reticulum isolated from the primate hearts revealed marked changes in calcium transport activities. It is concluded that the marked depression in cardiac sarcoplasmic reticulum functions indicates altered calcium homeostasis in the casted-primate heart which could be a factor in cardiovascular deconditioning.
Inside-outside distribution and diffusion of phosphatidylcholine in rat sarcoplasmic reticulum as determined by 13C NMR and phosphatidylcholine exchange protein
Kruijff, B. de; Besselaar, A.M.H.P. van den; Bosch, H. van den; Deenen, L.L.M. van
1. 1. The transverse distribution of phosphatidylcholine in rat sarcoplasmic reticulum was investigated employing 13C NMR in conjunction with the shift reagent DyCl3. 2. 2. Sarcoplasmic reticulum phosphatidylcholine was enriched with 13C by feeding rats a diet containing [N-Me3-13C]choline. Up to
Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats
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V.A. Vizotto
Full Text Available We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2, phospholamban (PLB, and ryanodine channel (RYR2 mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01. The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively. Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.
Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle
DEFF Research Database (Denmark)
Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs
The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate...... to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which.......05). Despite a slowing of the relaxation rate, we did not find any significant alterations in the SR Ca(2+) uptake function. These data demonstrate that the Ca(2+) release mechanism of SR is sensitive to repetitive in vitro muscle contraction. Moreover, the results indicate that +dF/dt to some extent depends...
Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I
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Campbell, Kevin Peter [Univ. of Rochester, NY (United States)
Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.
Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.
Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica
Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression. Copyright © 2011 Elsevier Inc. All rights reserved.
[Effect of calmodulin and 3':5'-AMP-dependent protein kinases on calcium transport by sarcoplasmic reticulum of normal rabbit myocardium and in toxico-allergic myocarditis].
Karsanov, N V; Khugashvili, Z G
It was demonstrated that under normal conditions calmodulin and exogenous 3':5'-AMP-dependent protein kinase considerably active Ca2+ transport by sarcoplasmic reticulum of rabbit myocardium; a combined action of these compounds produces an additive effect. The protein-inhibitor of 3':5'-AMP-dependent protein kinase and trifluoroperazine eliminate the activating effect of 3':5'-AMP-dependent protein kinase; in addition, trifluoroperazine decreases significantly the basal level of Ca2+ uptake. The 3':5'-AMP-dependent activation of Ca2+ transport becomes apparent after Ca2+-calmodulin-dependent phosphorylation of FSR membrane proteins. In toxico-allergic myocarditis calmodulin and 3':5'-AMP-dependent protein kinase do not activate the low level of Ca2+ uptake. No differences were observed between the action of calmodulin and 3':5'-AMP-dependent protein kinase isolated from normal and pathological rabbit heart. A conclusion is drawn that the decrease of Ca2+ transport is due to the impairment of Ca2+-calmodulin and 3':5'-AMP-dependent phosphorylation in sarcoplasmic reticulum membranes.
Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.
Simon, B J; Klein, M G; Schneider, M F
The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of
Effects of caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle fibres.
Klein, M G; Simon, B J; Schneider, M F
1. Resting myoplasmic [Ca2+] and [Ca2+] transients (delta [Ca2+]) were monitored using Fura-2 fluorescence and Antipyrylazo III absorbance signals from voltage-clamped segments of cut frog skeletal muscle fibres in the presence and absence of 0.5 mM-caffeine. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from delta [Ca2+]. 2. delta [Ca2+] and Rrel were increased in caffeine for all pulses. The decline of delta [Ca2+] was slower after a given pulse in caffeine than without caffeine. Resting [Ca2+] was slightly elevated in caffeine. 3. The voltage dependence of the peak value of Rrel and of the steady level of Rrel at the end of a 60-120 ms pulse were both shifted towards more negative voltages in caffeine. For relatively small pulses the voltage at which a given release waveform was observed was also shifted to more negative voltages. 4. Intramembrane charge movements measured in the same fibres in which the above changes in Rrel were observed showed no significant changes in caffeine. 5. In caffeine calcium release continued for many milliseconds after the end of a short (10 ms) pulse. Continued release after a pulse was not observed without caffeine and was probably due to positive feedback of elevated [Ca2+] on calcium release resulting from calcium-induced calcium release in caffeine. 6. Intramembrane charge movements after short pulses showed no change in caffeine that could account for the continued calcium release after the pulse. 7. Continued release after short pulses in caffeine decreased as the pulse duration was increased and was absent for pulses of 60 ms or longer. Rrel also inactivated during such pulses. 8. Relatively large and long conditioning pulses in caffeine suppressed both the peak Rrel and the continued release after short pulses. Peak release and continued release after short pulses recovered in parallel with increasing recovery time following suppression by a conditioning pulse in caffeine. 9. These
N-acetylcysteine protects against bupivacaine-induced myotoxicity caused by oxidative and sarcoplasmic reticulum stress in human skeletal myotubes.
Galbes, Olivier; Bourret, Annick; Nouette-Gaulain, Karine; Pillard, Fabien; Matecki, Stefan; Py, Guillaume; Mercier, Jacques; Capdevila, Xavier; Philips, Alexandre
Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain. Here, the authors investigate the mechanisms of local anesthetic-induced myotoxicity and assess the protective effect of N-acetylcysteine. The authors used primary cell cultures of human skeletal muscle myoblasts to study local anesthetic adverse effects. Production of reactive oxygen species was investigated in human skeletal myotubes by fluorescence microscopy. Expression of sarcoplasmic/endoplasmic reticulum stress markers and induction of apoptosis were followed by immunofluorescence and Western blot analysis. Finally, the effect of N-acetylcysteine on bupivacaine-induced myotoxicity was investigated in vitro. Bupivacaine sequentially induced reactive oxygen species production, oxidative stress, sarcoplasmic/endoplasmic reticulum stress, and activation of caspases 9 and 7 in human differentiated myoblasts. These iatrogenic effects were prevented by N-acetylcysteine. The authors demonstrated a protective effect of N-acetylcysteine against bupivacaine-induced sarcoplasmic/endoplasmic reticulum stress and apoptosis in primary human skeletal muscle cell.
Time course of activation of calcium release from sarcoplasmic reticulum in skeletal muscle.
Simon, B J; Schneider, M F
Myoplasmic free calcium transients were measured with antipyrylazo III in voltage clamped segments of frog skeletal muscle fibers and were used to calculate the rate of release (Rrel) of calcium from the sarcoplasmic reticulum. Intramembrane charge movement was measured for the same pulses in the same fibers. During a depolarizing pulse Rrel rose to an early peak and then decayed relatively rapidly but incompletely due to calcium-dependent inactivation (Schneider M.F., and B.J. Simon. 1988. J. Physiol. (Lond.). 405:727-745). Two approaches were used to determine release activation independent of the effects of inactivation: (a) a mathematical correction based on the assumption that inactivation was a process occurring in parallel with and independently of activation; (b) an experimental procedure in which release was maximally inactivated by a large short prepulse and then the remaining noninactivatable component of release was monitored during a subsequent test pulse. Both procedures gave the same time course of activation of release. Release activation paralleled the time course of intramembrane charge movement but was delayed by a few milliseconds.
Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence.
Takara, Delia; Sánchez, Gabriel A; Toma, Augusto F; Bonazzola, Patricia; Alonso, Guillermo L
Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca(2+) uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.
Protein kinase C inhibits Ca sup 2+ accumulation in cardiac sarcoplasmic reticulum
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Rogers, T.B.; Gaa, S.T.; Massey, C.; Doesemeci, A. (Univ. of Maryland School of Medicine, Baltimore (USA))
It is now recognized that phorbol esters are negative inotropic agents in mammalian heart which presumably act via stimulation of Ca2(+)-activated phospholipid-dependent protein kinase (PKC). The goal in the present study was to identify the underlying cellular processes. Digitonin-permeabilized cultured neonatal rat ventricular myocytes were used to study biochemical and functional effects of phorbol esters on cardiac sarcoplasmic reticulum (SR). These cells contracted spontaneously at 3 microM Ca2+. Beating was inhibited by 10 microM ryanodine and was insensitive to 1 microM nifedipine. Thus, beating behavior results from the phasic oscillation of Ca2+ transport by SR in this preparation. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreased frequency by 30%, suggesting that Ca2+ transport by SR had been reduced. Whereas cAMP stimulated the rate of oxalate-supported 45Ca2+ uptake 2-fold, phorbol esters, TPA, and phorbol 12,13-dibutyrate inhibited this process by about 45%. The effects of phorbols were specific: (a) the alpha-analogues of TPA and phorbol 12,13-dibutyrate were inactive; and (b) the phorbol esters had no effect on Ca2+ transport in cells that had been depleted of PKC. TPA decreased oxalate-stimulated Ca2+ uptake over the entire range of Ca2+ concentrations, from 0.1 to 10 microM, by at least 70% without shifting the half-maximal effective Ca2+ concentration. Taken together these results indicate that the effects of phorbol ester on cardiac contraction are due to decreased Ca2+ transport by the SR and that these responses are mediated by PKC. These studies support the interpretation that the negative inotropic effects of phorbol esters are due, in part, to decreased SR function.
Expression of sarcoplasmic-endoplasmic reticulum Ca-ATPase isoforms in masticatory muscles.
Sánchez, Gabriel A; Trinks, Pablo W; Richard, Susana B; Di Croce, Daniel E; Takara, Delia
The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport. © 2013 Eur J Oral Sci.
Variable luminal sarcoplasmic reticulum Ca(2+) buffer capacity in smooth muscle cells.
Dagnino-Acosta, Adán; Guerrero-Hernández, Agustín
Sarcoplasmic reticulum contains the internal Ca(2+) store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca(2+) level is the same all throughout the SR; however, whether the Ca(2+) buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca(2+) buffer capacity of the SR by comparing the reduction in SR Ca(2+) levels with the corresponding increase in [Ca(2+)](i) during activation of either IP(3)Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca(2+) buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca(2+) buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca(2+) buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca(2+) replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca(2+) influx, indicate the relevance of luminal SR Ca(2+) buffer capacity in the [Ca(2+)](i) response. To further study the importance of luminal SR Ca(2+) buffer capacity in the release process we used low levels of heparin to partially inhibit IP(3)Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca(2+) levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca(2+)-binding proteins in the high-capacity, low-affinity conformation, particularly for IP(3)R-mediated Ca(2+) release.
Sarcoplasmic reticulum Ca(2+)-pump dysfunction in rat cardiomyocytes briefly exposed to hydroxyl radicals.
Morris, T E; Sulakhe, P V
The effects of hydroxyl radical exposure of intact cardiomyocytes on sarcoplasmic reticulum (SR) function were investigated. For this purpose, isolated rat heart myocytes were exposed briefly (1 min) to the hydroxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobis-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and following this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to either the hydroxyl radical-generating system or DTNB. The exposure of myocytes to the hydroxyl radical, but not DTNB, markedly increased the amount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [3H]-NEM binding to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and catalase were found to offer complete protection against the inhibitory effect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activity. In the SR isolated from the treated myocytes, the stimulatory effects of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTNB on the functioning of SR occurs rapidly in the intact cardiomyocytes. The hydroxyl radical-provoked damage involves both protein sulfhydryl and lipid oxidation.
Cysteine-674 oxidation and degradation of sarcoplasmic reticulum Ca(2+) ATPase in diabetic pig aorta.
Ying, Jia; Sharov, Victor; Xu, Shanqin; Jiang, Bingbing; Gerrity, Ross; Schöneich, Christian; Cohen, Richard A
The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.
Sarcoplasmic reticulum buffering of myoplasmic calcium in bovine coronary artery smooth muscle.
Sturek, M; Kunda, K; Hu, Q
1. We tested the hypothesis that the sarcoplasmic reticulum (SR) buffers (attenuates) the increase in averaged myoplasmic free [Ca2+] (Ca(im)) resulting from Ca2+ influx. 2. Fura-2 measurements of Ca(im) were obtained in single smooth muscle cells freshly dispersed from bovine coronary artery. 3. Caffeine (5 x 10(-3) M) elicited a transient increase in Ca(im) and depleted the SR Ca2+ store. In the continued presence of caffeine or 10(-5) M-ryanodine SR buffering of Ca(im) was inhibited. Subsequent exposure to high extracellular [K+] (greater than 30 mM, equimolar Na+ removal) elicited a 2-fold more rapid and 2-fold greater peak increase in Ca(im) than high K+ elicited when SR buffering of Ca(im) was normal. The augmented increase in Ca(im) was inhibited 35% by 10(-5) M-diltiazem, 65% by 2 x 10(-4) M-LaCl3, and 87% in Ca(2+)-free external solution. 4. When Ca(im) buffering capacity was increased by partially depleting the SR with a transient (1 min) exposure to caffeine, subsequent exposure to 80 nM-K+ solution increased Ca(im) almost 2-fold more slowly than 80 mM-K+ before depletion of Ca2+ from the SR. However, the influxing Ca2+ was sequestered by the SR and refilled it, as evident by the subsequent caffeine-induced Ca(im) transient being identical to the first. Increasing extracellular [K+] (thus, increasing depolarization and Na+ removal) caused proportional increases in Ca(im) and the subsequent caffeine-induced Ca(im) transients were proportionally larger, indicating a graded filling of the SR by Ca2+ influx. 5. Diltiazem (10(-5) M) inhibited the refilling of the SR achieved by 80 mM-K+, by 26%. Refilling was inhibited 76% by 80 mM-K+, Ca(2+)-free solution, indicating the fraction of refilling dependent on influx of Ca2+ through voltage-gated Ca2+ channels, leak channels, and other influx pathways. Mild depolarization with 35 mM-K+ (no Na+ removal) often caused no increase in Ca(im), but influx through voltage-gated Ca2+ channels occurred because the SR Ca2
Modulation of sarcoplasmic reticulum Ca2+ release by glycolysis in cat atrial myocytes.
Kockskämper, Jens; Zima, Aleksey V; Blatter, Lothar A
In cardiac myocytes, glycolysis and excitation-contraction (E-C) coupling are functionally coupled. We studied the effects of inhibitors (2-deoxy-D-glucose (2-DG), iodoacetate (IAA)), intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP)) and products (pyruvate, L-lactate) of glycolysis on sarcoplasmic reticulum (SR) Ca(2+) release and uptake in intact and permeabilized cat atrial myocytes. In field-stimulated (0.5-0.7 Hz) intact myocytes, 2-DG (10 mm) and IAA (1 mm) caused elevation of diastolic [Ca(2+)](i) and [Ca(2+)](i) transient alternans (Ca(2+) alternans) followed by a decrease of the amplitude of the [Ca(2+)](i) transient. Focal application of 2-DG resulted in local Ca(2+) alternans that was confined to the region of exposure. 2-DG and IAA slowed the decay kinetics of the [Ca(2+)](i) transient and delayed its recovery (positive staircase) after complete SR depletion, suggesting impaired activity of the SR Ca(2+)-ATPase (SERCA). 2-DG and IAA reduced the rate of reuptake of Ca(2+) into the SR which was accompanied by a 15-20% decrease of SR Ca(2+) load. Major changes of mitochondrial redox state (measured as FAD autofluorescence) were not observed after inhibition of glycolysis. Pyruvate (10 mm) and L-lactate (10 mm) elicited similar changes of the [Ca(2+)](i) transient. Pyruvate, L-lactate and IAA - but not 2-DG - induced intracellular acidosis. Recording of single channel activity of ryanodine receptors (RyRs) incorporated into lipid bilayers revealed complex modulation by glycolytic intermediates and products (1 mm each): some were without effect (G6P, PEP, L-lactate) while others either increased (F6P, +40%; FBP, +265%) or decreased (pyruvate, -58%) the open probability of the RyR. Consistent with these findings, spontaneous SR Ca(2+) release (Ca(2+) sparks) in permeabilized myocytes was facilitated by FBP and inhibited by pyruvate. The results indicate that in atrial myocytes
Activity of Ca(2+,Mg(2+-ATPase of sarcoplasmic reticulum and contraction strength of the frog skeletal muscles under the effect of organophosphorus insecticides
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D. M. Nozdrenko
Full Text Available The results of an experimental study of organophosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+,Mg2+-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+,Mg2+-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.
Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.
Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G
The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase
Study of the function of sarcoplasmic reticulum of vascular smooth muscle during activation due to depolarization-induced calcium influx
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Hwang, K.S.
The role of sarcoplasmic reticulum (SR) in vascular smooth muscle was evaluated with respect to regulation of myoplasmic Ca{sup 2+} during the Ca{sup 2+} entry induced by depolarization. Calcium agonist, Bay K8644, stimulated Ca{sup 2+} influx as well as tension in physiological salt solution, (PSS) in contrast to the priming effects due to the depolarization originally reported. Disparity, however, was found between the Ca{sup 2+} entered and tension developed. Correlation between the tension and {sup 45}Ca influx showed a typical threshold phenomenon; the basal Ca{sup 2+} influx can be raised to a certain level (25%) without tension induction, after which a minor increase in Ca{sup 2+} influx produced significant tension. This subthreshold Ca{sup 2+} influx was found accumulated in the caffeine-sensitive Ca stores, the SR. This confirmed the dependency of tension on the rate of Ca{sup 2+} entry demonstrated by a previous report.
Nandrolone decanoate treatment affects sarcoplasmic reticulum Ca(2+) ATPase function in skinned rat slow- and fast-twitch fibres.
Bouhlel, Aicha; Joumaa, Wissam H; Léoty, Claude
The effects of anabolic-androgenic steroid administration on the function of the sarcoplasmic reticulum (SR) pump were investigated in chemically skinned fibres from the extensor digitorum longus (EDL) and soleus muscles of sedentary rats. Twenty male rats were divided into two groups, one group received an intramuscular injection of nandrolone decanoate (15 mg x kg(-1)) weekly for 8 weeks, the second received similar weekly doses of vehicle (sterile peanut oil). Compared with control muscles, nandrolone decanoate treatment reduced SR Ca(2+) loading in EDL and soleus fibres by 49% and 29%, respectively. In control and treated muscles, the rate of Ca(2+) leakage depended on the quantity of Ca(2+) loaded. Furthermore, for similar SR Ca(2+) contents, the Ca(2+) leakage rate was not significantly modified by nandrolone decanoate treatment. Nandrolone decanoate treatment thus affects Ca (2+) uptake by the SR in a fibre-type dependent manner.
Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation
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Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.; Opresko, Lee; Mayer, M. Uljana; Brady, Scott T.; Squier, Thomas C.; Bigelow, Diana J.
The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, and YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.
Post mortem changes in Ca2+ transporting proteins of sarcoplasmic reticulum in dependence on malignant hyperthermia status in pigs.
Küchenmeister, U; Kuhn, G; Wegner, J; Nürnberg, G; Ender, K
Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined. The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2
ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.
Kourie, J I
Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl- channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl- channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, Imax and integral current, I') and kinetic parameters (frequency of opening FO, probability of the channel being open PO, mean open TO and closed Tc times) show a nonspecific block of the voltage- and Ca2+-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca2+ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg2+. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP4- > ADP3- > > > AMP2-. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP]cis and [Ca2+]cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca2+ between the SR and the sarcoplasm.
Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.
Haustein, Moritz; Hannes, Tobias; Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus
Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, pmyocardium (n=5, pmyocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.
Involvement of sarcoplasmic reticulum 'Ca2+ release channels' in excitation-contraction coupling in vertebrate skeletal muscle.
Brunder, D G; Györke, S; Dettbarn, C; Palade, P
1. Pharmacological blockers of calcium-induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation-contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9-aminoacridine inhibited the SR Ca2+ release associated with excitation-contraction (E-C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca2+ uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation-contraction coupling than to block Ca2+ release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR 'Ca2+ release channels' (the ryanodine receptors that mediate Ca(2+)-induced Ca2+ release), the equal effectiveness of these blockers at inhibiting excitation-contraction (E-C) coupling and its potentiation by caffeine suggests that the SR 'Ca2+ release channels' are indeed involved in excitation-concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E-C coupling. PMID:1380087
High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart.
Aston, Daniel; Capel, Rebecca A; Ford, Kerrie L; Christian, Helen C; Mirams, Gary R; Rog-Zielinska, Eva A; Kohl, Peter; Galione, Antony; Burton, Rebecca A B; Terrar, Derek A
Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.
Effects of oestrogen on sarcoplasmic reticulum Ca2+-ATPase activity and gene expression in genioglossus in chronic intermittent hypoxia rat.
Liu, Yue-Hua; Li, Wen; Song, Wei-Hua
This study was designed to investigate the effects of oestrogen on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and gene expression in ovariectomised rats under the condition of chronic intermittent hypoxia (CIH). Thirty-two female Sprague-Dawley rats were randomly divided into four groups: the normal control group (NC), the CIH group (CIH), the CIH-ovariectomised group (CIH+OVX), and the group of CIH-ovariectomised rats receiving estradiol replacement (CIH+OVX+E(2)). Rats in the latter three groups were exposed to CIH for 5 weeks. The animals were killed before genioglossus (GG) was rapidly excised, and their body and uterus mass were determined. Estradiol level was detected by radioimmunoassay. SR Ca(2+)-ATPase (SERCA) activity was observed by detecting inorganic phosphorus ion, and the SERCA mRNA level was measured using real-time quantitative polymerase chain reaction (real-time PCR). It was found that, compared with the NC group, the SERCA activity and mRNA level were remarkably reduced (pSERCA activity and mRNA level were also significantly reduced (pSERCA activity and mRNA level significantly increased (pSERCA activity and mRNA expression, and oestrogen-deficiency could exacerbate this effect; whilst estradiol replacement can partially reverse the effect of CIH in ovariectomised rats.
Interactions of Phosphate Groups of ATP and Aspartyl Phosphate with the Sarcoplasmic Reticulum Ca2+-ATPase: An FTIR Study
Liu, Man; Krasteva, Maria; Barth, Andreas
Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([β-18O2, βγ-18O]ATP and [γ-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm−1 are affected by labeling of the β-phosphate, bands likely near 1154, and 1098–1089 cm−1 are affected by γ-phosphate labeling. The findings indicate that the strength of interactions of β- and γ- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm−1, were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water. PMID:16169973
Pycnogenol® and Ginkgo biloba extract: effect on peroxynitrite-oxidized sarcoplasmic reticulum Ca2+-ATPase
Žižková, Petronela; Viskupičová, Jana; Horáková, L'ubica
The effect of two natural standardized plant extracts, Pycnogenol® and EGb 761, on sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and posttranslational modifications induced by peroxynitrite was investigated to assess their possible protective role. EGb 761 was found to have a protective effect on SERCA activity in the concentration range of 5–40 µg/ml. On the other hand, Pycnogenol® caused a decrease of SERCA activity at concentrations of 25 µg/ml. EGb 761 did not prevent protein carbonyl formation upon oxidation with peroxynitrite. On the contrary, Pycnogenol® at the concentrations of 5 and 10 µg/ml significantly decreased the level of protein carbonyls by 44% and 54%, respectively. Neither Pycnogenol® nor EGb 761 exerted a protective effect against thiol group oxidation.The plant extracts studied modulated peroxynitrite-injured SERCA activity by different ways and failed to correlate with posttranslational modifications. Their effect seems to be associated with their ability to change SERCA conformation rather than by their antioxidant capacity. PMID:21331179
Ca(2+) leakage out of the sarcoplasmic reticulum is increased in type I skeletal muscle fibres in aged humans.
Lamboley, C R; Wyckelsma, V L; McKenna, M J; Murphy, R M; Lamb, G D
The amount of Ca(2+) stored in the sarcoplasmic reticulum (SR) of muscle fibres is decreased in aged individuals, and an important question is whether this results from increased Ca(2+) leakage out through the Ca(2+) release channels (ryanodine receptors; RyRs). The present study examined the effects of blocking the RyRs with Mg(2+), or applying a strong reducing treatment, on net Ca(2+) accumulation by the SR in skinned muscle fibres from Old (∼70 years) and Young (∼24 years) adults. Raising cytoplasmic [Mg(2+)] and reducing treatment increased net SR Ca(2+) accumulation in type I fibres of Old subjects relative to that in Young. The densities of RyRs and dihydropyridine receptors were not significantly changed in the muscle of Old subjects. These findings indicate that oxidative modification of the RyRs causes increased Ca(2+) leakage from the SR in muscle fibres in Old subjects, which probably deleteriously affects normal muscle function both directly and indirectly. The present study examined whether the lower Ca(2+) storage levels in the sarcoplasmic reticulum (SR) in vastus lateralis muscle fibres in Old (70 ± 4 years) relative to Young (24 ± 4 years) human subjects is the result of increased leakage of Ca(2+) out of the SR through the Ca(2+) release channels/ryanodine receptors (RyRs) and due to oxidative modification of the RyRs. SR Ca(2+) accumulation in mechanically skinned muscle fibres was examined in the presence of 1, 3 or 10 mm cytoplasmic Mg(2+) because raising [Mg(2+)] strongly inhibits Ca(2+) efflux through the RyRs. In type I fibres of Old subjects, SR Ca(2+) accumulation in the presence of 1 mm Mg(2+) approached saturation at shorter loading times than in Young subjects, consistent with Ca(2+) leakage limiting net uptake, and raising [Mg(2+)] to 10 mm in such fibres increased maximal SR Ca(2+) accumulation. No significant differences were seen in type II fibres. Treatment with dithiothreitol (10 mm for 5 min), a strong reducing agent, also
Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres.
Lamboley, C R; Murphy, R M; McKenna, M J; Lamb, G D
The relationship between sarcoplasmic reticulum (SR) Ca(2+) content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca(2+) content and maximal Ca(2+) capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca(2+) content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca(2+) per litre of fibre, respectively), with virtually all of this Ca(2+) evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg(2+)] solution (only 0.08 ± 0.01 and fibres, respectively (P fibres, where the SR remained functional, repeated cycles of caffeine-induced Ca(2+) release and subsequent Ca(2+) reloading similarly indicated that (i) maximal SR Ca(2+) content was lower in type I fibres than in type II fibres (P fibres compared to type II fibres (∼59% and 41%, respectively, P fibres were found on average to contain ∼3-fold more CSQ1 and ∼5-fold less CSQ2 than type I fibres (P fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.
Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.
Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan
High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.
Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation
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Bassani R.A.
Full Text Available Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A, Na+-Ca2+ exchanger (NCX and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake. To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW. Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM, whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM. The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.
Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?
Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc
We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.
Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.
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Mani Sadredini
Full Text Available Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial
Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.
Peter, Angela K; Miller, Gaynor; Capote, Joana; DiFranco, Marino; Solares-Pérez, Alhondra; Wang, Emily L; Heighway, Jim; Coral-Vázquez, Ramón M; Vergara, Julio; Crosbie-Watson, Rachelle H
Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd-/-) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. Alternative splicing of proteins from the SG
Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.
Peter, Angela K; Miller, Gaynor; Capote, Joana; DiFranco, Marino; Solares-Pérez, Alhondra; Wang, Emily L; Heighway, Jim; Coral-Vázquez, Ramón M; Vergara, Julio; Crosbie-Watson, Rachelle H
Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd-/-) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. Alternative splicing of proteins from the SG
Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells.
Fabiato, A; Fabiato, F
1. Fragments of single cardiac cells were obtained by homogenization of ventricular tissue from adult rats. Remaining pieces of sacrolemma were removed by micro-dissection. Tension was recorded from the ends of the skinned (sarcolemma-free) cells with a photodiode force transducer. 2. In the presence of a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a tonic tension was obtained that increased according to t sigmoid curve when the free ([Ca2+] was increased from 10(-6-75)M to 10(-5-0)M. This curve was not modified by the destruction of the sarcoplasmic reticulum (SR) by the detergent Brij 58. Therefore, the tonic tension corresponded to the direct effect of the free [Ca2+] present in the buffer on the myofilaments. 3. In the presence of a slight buffering of the free [Ca2+] with 0-050 mM total EGTA, cyclic contractions were observed that were attributed to cyclic releases and re-sequestrations of Ca2+ by the SR. The absence of effect of azide and ruthenium red on the cyclic contractions obtained at a free [Ca2+] lower than 10(-6-50)M demonstrated that the mitochondria played no role in the triggering of these contractions. 4. Cyclic contractions were induced by a slight variation of free [Ca2+] in the buffer from 10(-7-65)M to 10(-7-40)M. Their amplitude at 10(-7-40)M free Ca2+ was equal to the tonic tension developed by a free [Ca2+] 20 times higher applied to the myofilaments when the SR was destroyed by detergent or functionally inhibited by high total [EGTA]. It was concluded that these cyclic contractions corresponded to a Ca2+-triggered release of Ca2+ from the SR. 5. The cyclic contractions were induced by the filling of the SR with Ca2+ to a critical level at which it released a fraction of the Ca2+ it contained. Each contraction was followed by a re-sequestration of Ca2+, the kinetics of which conditioned the duration of the cycles. 6. The amplitude of the cyclic contractions increased when the free [Ca2+] that triggered them was increased
Puerarin Enhances Ca2+ Reuptake and Ca2+ Content of Sarcoplasmic Reticulum in Murine Embryonic Stem Cell-Derived Cardiomyocytes via Upregulation of SERCA2a
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Lu Wang
Full Text Available Background/Aims: The embryonic stem cell-derived cardiomyocytes (ES-CMs serve as potential sources for cardiac regenerative therapy. However, the immature sarcoplasmic reticulum (SR function of ES-CMs prevents its application. In this report, we examined the effect of puerarin, an isoflavone compound, on SR function of murine ES-CMs. Methods: Murine ES-CMs were harvested by embryoid body-based differentiation method. Confocal calcium imaging and whole-cell patch clamps were performed to assess the function of SR. The mRNA expression levels of SR-related genes were examined by quantitative PCR. The protein expression of sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a was evaluated by immunofluorescent and western blot. Results: Long-term application of puerarin promotes basic properties of spontaneous calcium transient with increased amplitude, decay velocity, and decreased duration. Puerarin fails to alter ICa,L but increases the Ca2+ content of SR. Puerarin-treated ES-CMs have intact SR Ca2+ cycling with more SR Ca2+ reuptake. Long-term application of puerarin asynchronously upregulates the mRNA and protein expression of SERCA2a, as well as the transcripts of calsequestrin and triadin in developing ES-CMs. Application of puerarin during the stage of post-cardiac differentiation upregulates dose-dependently the transcripts of SERCA2a, phospholamban and tridin which can be reversed by the inhibitors of the PI3K/Akt and MAPK/ERK signaling pathways, but shows no effect on the protein expression of SERCA2a. Conclusion: This study demonstrates that long-term puerarin treatment enhances Ca2+ reuptake and Ca2+ content via upregulation of SERCA2a.
Constitutive cardiac overexpression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase delays myocardial failure after myocardial infarction in rats at a cost of increased acute arrhythmias.
Chen, Ying; Escoubet, Brigitte; Prunier, Fabrice; Amour, Julien; Simonides, Warner S; Vivien, Benoît; Lenoir, Christophe; Heimburger, Michèle; Choqueux, Christine; Gellen, Barnabas; Riou, Bruno; Michel, Jean-Baptiste; Franz, Wolfgang M; Mercadier, Jean-Jacques
Heart failure often complicates myocardial infarction (MI), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is underexpressed in the failing myocardium. We examined the effect of preexisting cardiac SERCA2a protein overexpression on rat survival and left ventricular (LV) remodeling after MI. Baseline myocardial SERCA2a expression was 37% higher in transgenic (TG) rats than in their wild-type (WT) controls, consistent with enhanced myocardial function. The mortality rate of TG rats during the 24 hours after surgical MI was higher than that of WT rats (71% versus 35%, P<0.001), associated with a higher frequency of ventricular arrhythmias, and was normalized by lidocaine treatment. The increased acute-phase mortality in TG rats was not accompanied by increased 6-month mortality. Function of the noninfarcted myocardium, as assessed by tissue Doppler imaging, was higher in TG rats than in WT rats for up to 1 month after MI, a beneficial effect no longer observed at 3 months. LV remodeling and global function were similar in TG and WT rats. No difference in papillary muscle function was found at 6 months. Constitutive cardiac SERCA2a overexpression has a transient beneficial effect on remote myocardium function in rat MI, with no improvement in LV global function or prevention of LV remodeling and failure. This benefit is associated with a higher risk of acute mortality, which is prevented by lidocaine treatment.
Fluvastatin and atorvastatin affect calcium homeostasis of rat skeletal muscle fibers in vivo and in vitro by impairing the sarcoplasmic reticulum/mitochondria Ca2+-release system.
Liantonio, Antonella; Giannuzzi, Viviana; Cippone, Valentina; Camerino, Giulia Maria; Pierno, Sabata; Camerino, Diana Conte
The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg-1) and atorvastatin (5 and 10 mg kg-1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca2+]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca2+]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca2+]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca2+]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statin-induced Ca2+ release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca2+ release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drugs.
Changes in T-Tubules and Sarcoplasmic Reticulum in Ventricular Myocytes in Early Cardiac Hypertrophy in a Pressure Overload Rat Model
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Perla Pérez-Treviño
Full Text Available Background/Aims: Pressure-overload (PO causes cardiac hypertrophy (CH, and eventually leads to heart failure (HF. HF ventricular myocytes present transverse-tubules (TT loss or disarrangement and decreased sarcoplasmic reticulum (SR density, and both contribute to altered Ca2+ signaling and heart dysfunction. It has been shown that TT remodeling precedes HF, however, it is unknown whether SR structural and functional remodeling also starts early in CH. Methods: Using confocal microscopy, we assessed TT (with Di-8-ANNEPS and SR (with SR-trapped Mag-Fluo-4 densities, as well as SR fluorophore diffusion (fluorescence recovery after photobleach; FRAP, cytosolic Ca2+ signaling and ex vivo cardiac performance in a PO rat hypertrophy model induced by abdominal aortic constriction (at 6 weeks. Results: Rats developed CH, while cardiac performance, basal and upon β-adrenergic stimulation, remained unaltered. TT density decreased by ∼14%, without spatial disarrangement, while SR density decreased by ∼7%. More important, FRAP was ∼30% slower, but with similar maximum recovery, suggesting decreased SR interconnectivity. Systolic and diastolic Ca2+ signaling and SR Ca2+ content were unaltered. Conclusion: SR remodeling is an early CH event, similar to TT remodeling, appearing during compensated hypertrophy. Nevertheless, myocytes can withstand those moderate structural changes in SR and TT, preserving normal Ca2+ signaling and contractility.
Homotypic fusion of endoplasmic reticulum membranes in plant cells
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Junjie eHu
Full Text Available The endoplasmic reticulum (ER is a membrane-bounded organelle whose membrane comprises a network of tubules and sheets. The formation of these characteristic shapes and maintenance of their continuity through homotypic membrane fusion appears to be critical for the proper functioning of the ER. The atlastins (ATLs, a family of ER-localized dynamin-like GTPases, have been identified as fusogens of the ER membranes in metazoans. Mutations of the ATL proteins in mammalian cells cause morphological defects in the ER, and purified Drosophila ATL mediates membrane fusion in vitro. Plant cells do not possess ATL, but a family of similar GTPases, named root hair defective 3 (RHD3, are likely the functional orthologs of ATLs. In this review, we summarize recent advances in our understanding of how RHD3 proteins play a role in homotypic ER fusion. We also discuss the possible physiological significance of forming a tubular ER network in plant cells.
A pK change of acidic residues contributes to cation countertransport in the Ca-ATPase of sarcoplasmic reticulum. Role of H+ in Ca(2+)-ATPase countertransport.
Yu, X; Hao, L; Inesi, G
Proteoliposomal vesicles reconstituted with sarcoplasmic reticulum ATPase and exogenous lipids sustain ATP-dependent Ca2+ uptake and H+ ejection, as well as net charge displacement by Ca2+. We have studied the effect of lumenal (inner) and medium (extravesicular) pH variations on the countertransport ratios of H+ and Ca2+. We find that the Ca2+/H+ molar ratio is approximately 1 when the lumenal and medium pH is near neutrality, but changes with a specific pattern when the medium pH is varied in the presence of a constant lumenal pH and when the lumenal pH is varied in the presence of a constant medium pH. Empirical analysis of the experimental data shows that the apparent pK of the residue(s) releasing H+ into the medium is approximately 6.1, whereas the apparent pK of the residue(s) binding lumenal H+ is approximately 7.7. Assuming that the same acidic residues are involved in H+ and Ca2+ countertransport, our findings suggest a lower affinity for H+ in their outward orientation (prevalent in the ground state of the enzyme) and a higher affinity for H+ in lumenal orientation (prevalent in the phosphorylated state of the enzyme). Cyclic pK changes, coupled to ATP utilization, promote cation exchange, Ca2+ uptake, and H+ ejection by the vesicles. The stoichiometry of countertransport and net charge displacement is matched by a corresponding electrogenic behavior. A calculation of voltage development related to initial rates of charge transfer (dV/dt = (dQ/dt)/Cm) is given as a corrective replacement of a previous steady state calculation.
Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function.
Provence, Aaron; Rovner, Eric S; Petkov, Georgi V
We recently reported key physiologic roles for Ca(2+)-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca(2+)-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca(2+), inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release from the sarcoplasmic reticulum represents a potential Ca(2+) source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca(2+) release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca(2+)-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca(2+)-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM.
Tissue-specific expression of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) 1, 2, 3 during Xenopus laevis development.
Pegoraro, Caterina; Pollet, Nicolas; Monsoro-Burq, Anne H
Calcium-ATPase pumps are critical in most cells, to sequester calcium into intracytoplasmic stores and regulate general calcium signalling. In addition, cell-specific needs for calcium signals have been described and employ a diversity of calcium ATPases in adult tissues and oocytes. A major family of such calcium pumps is ATP2A/SERCA family, for Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases. Although largely studied in adults, the developmental expression of the atp2a/serca genes remains unknown. Here, we provide genome organisation in Xenopuslaevis and tropicalis and phylogeny of atp2a/serca genes in craniates. We detail embryonic expression for the three X. laevis atp2a/serca genes. We found that the three atp2a/serca genes are strongly conserved among vertebrates and display complementary and tissue-specific expression in embryos. These expression patterns present variations when compared to the data reported in adults. Atp2a1/serca1 is expressed as soon as the end of gastrulation in a subset of the myod-positive cells, and later labels prospective slow muscle cells in the superficial part of the somite. In contrast atp2a2/serca2 is found in a larger subset of cells, but is not ubiquitous as reported in adults. Notably, atp2a2/serca2 is prominently expressed in the neural-related tissues, i.e. the neural plate, cement gland, but is excluded from premigratory neural crest. Finally, atp2a3/serca3 expression is restricted to the ectoderm throughout development. Copyright © 2010 Elsevier B.V. All rights reserved.
Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells.
Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie
Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1-3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.
Differential regulation of the fiber type-specific gene expression of the sarcoplasmic reticulum calcium-ATPase isoforms induced by exercise training.
Morissette, Marc P; Susser, Shanel E; Stammers, Andrew N; O'Hara, Kimberley A; Gardiner, Phillip F; Sheppard, Patricia; Moffatt, Teri L; Duhamel, Todd A
The regulatory role of adenosine monophosphate-activated protein kinase (AMPK)-α2 on sarcoplasmic reticulum calcium-ATPase (SERCA) 1a and SERCA2a in different skeletal muscle fiber types has yet to be elucidated. Sedentary (Sed) or exercise-trained (Ex) wild-type (WT) and AMPKα2-kinase dead (KD) transgenic mice, which overexpress a mutated and inactivated AMPKα2 subunit, were utilized to characterize how genotype or exercise training influenced the regulation of SERCA isoforms in gastrocnemius. As expected, both Sed and Ex KD mice had >40% lower AMPK phosphorylation and 30% lower SERCA1a protein than WT mice (P SERCA2a protein was not different among KD and WT mice. Exercise increased SERCA1a and SERCA2a protein content among WT and KD mice, compared with their Sed counterparts. Maximal SERCA activity was lower in KD mice, compared with WT. Total phospholamban protein was higher in KD mice than in WT and lower in Ex compared with Sed mice. Exercise training increased phospholamban Ser(16) phosphorylation in WT mice. Laser capture microdissection and quantitative PCR indicated that SERCA1a mRNA expression among type I fibers was not altered by genotype or exercise, but SERCA2a mRNA was increased 30-fold in WT+Ex, compared with WT+Sed. In contrast, the exercise-stimulated increase for SERCA2a mRNA was blunted in KD mice. Exercise upregulated SERCA1a and SERCA2a mRNA among type II fibers, but was not altered by genotype. Collectively, these data suggest that exercise differentially influences SERCA isoform expression in type I and type II fibers. Additionally, AMPKα2 influences the regulation of SERCA2a mRNA in type I skeletal muscle fibers following exercise training. Copyright © 2014 the American Physiological Society.
Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1
Glukhov, Alexey V.; Kalyanasundaram, Anuradha; Lou, Qing; Hage, Lori T.; Hansen, Brian J.; Belevych, Andriy E.; Mohler, Peter J.; Knollmann, Björn C.; Periasamy, Muthu; Györke, Sandor; Fedorov, Vadim V.
Aims Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. Methods and results In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca2+ cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2−/−) mice. Casq2−/− mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca2+ cycling, including abnormal Ca2+ release, periods of significantly elevated diastolic Ca2+ levels leading to pauses and unstable pacemaker rate. Importantly, Ca2+ cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca2+ transient upstrokes throughout the atrial pacemaker complex. Conclusions Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca2+ release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients. PMID:24216388
Biphasic contractions induced by milrinone at low temperature in ferret ventricular muscle: role of the sarcoplasmic reticulum and transmembrane calcium influx.
Malecot, C O; Bers, D M; Katzung, B G
The effects of milrinone were studied in ferret papillary muscle stimulated at various rates and temperatures from 23 degrees to 36 degrees C. In voltage-clamp experiments, 50 micrograms/ml (0.237 mM) milrinone induced a 2.1-fold increase in calcium current at 28 degrees or 36 degrees C. At 50 micrograms/ml, milrinone transiently increased contractility in all muscles at 28 degrees C, but its steady-state effect was either increased (+50%) or decreased (-24.7%) steady-state twitch amplitude. A negative inotropic effect always occurred below 27 degrees C. Milrinone decreased the total twitch duration and split the twitch into two components (P1 and P2) in the absence of any evidence of aberrant conduction. Increasing milrinone concentration from 50 to 300 micrograms/ml decreased P1 and increased P2. Ryanodine (100 mM) or caffeine (10 mM) suppressed P1. Contractions elicited after 30 seconds of rest were also biphasic in the presence of milrinone, but not in its absence. P2 of post-rest contraction was increased by caffeine or calcium (10 mM) and decreased by cobalt (2 mM) when drugs were applied at the beginning of the rest. Ryanodine and caffeine also suppressed P1 of post-rest contraction. The evidence suggests that P1 may be caused by Ca release from the sarcoplasmic reticulum and P2 by increased Ca influx during the action potential via the calcium channel. It is also suggested that P2 may be present under control conditions, but to a lesser extent, and masked by a large P1.
Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle.
Lamboley, C R; Murphy, R M; McKenna, M J; Lamb, G D
The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.
Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia
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Wei XIN
Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.
Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.
Wang, Pengwei; Hawes, Chris; Hussey, Patrick J
The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants. Copyright © 2016. Published by Elsevier Ltd.
A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.
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Tobias Gröβl
Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.
Contractile properties and sarcoplasmic reticulum calcium content in type I and type II skeletal muscle fibres in active aged humans.
Lamboley, C R; Wyckelsma, V L; Dutka, T L; McKenna, M J; Murphy, R M; Lamb, G D
Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres. This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults. The maximum level of force production (per unit cross-sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium. The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast- and slow-twitch muscle fibres in the Old subjects. These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres. This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca(2+) content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ∼17% and Ca(2+) sensitivity was also reduced (pCa50 decreased ∼0.05 pCa units) relative to that in Young. S-Glutathionylation of fast troponin I (TnIf ) markedly increased Ca(2+) sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca(2+) content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca(2+) by a combined caffeine and low Mg(2+) stimulus, whereas in fibres of Old the amount of non-releasable Ca(2+) was significantly increased (by > 12% of endogenous Ca(2+) content). Western
IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release.
Xi, Q.; Adebiyi, A.; Zhao, G.; Chapman, K.E.; Waters, C.M.; Hassid, A.; Jaggar, J.H.
Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP(3)). IP(3) is generally considered to elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic
Tuning the structural coupling between the transmembrane and cytoplasmic domains of phospholamban to control sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function.
Ha, Kim N; Gustavsson, Martin; Veglia, Gianluigi
Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), the integral membrane enzyme responsible for 70 % of the removal of Ca(2+) from the cytosol, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a critical role in cardiac disease, and targeting Ca(2+) transport has been demonstrated to be a promising avenue in treating conditions of heart failure. Here, we designed a series of new mutants able to tune SERCA function, targeting the loop sequence that connects the transmembrane and cytoplasmic helices of PLN. We found that a variable degree of loss of inhibition mutants is attainable by engineering glycine mutations along PLN's loop domain. Remarkably, a double glycine mutation results in a complete loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using nuclear magnetic resonance spectroscopy, we rationalized the effects of these mutations in terms of entropic control on PLN function, whose inhibitory function can be modulated by increasing its conformational dynamics. However, if PLN mutations go past a threshold set by the phosphorylated state, they break the structural coupling between the transmembrane and cytoplasmic domains, resulting in a species that behaves as the inhibitory transmembrane domain alone. These studies provide new potential candidates for gene therapy to reverse the effects of heart failure.
The overexpression of nuclear envelope protein Lap2β induces endoplasmic reticulum reorganisation via membrane stacking
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Ekaterina G. Volkova
Some nuclear envelope proteins are localised to both the nuclear envelope and the endoplasmic reticulum; therefore, it seems plausible that even small amounts of these proteins can influence the organisation of the endoplasmic reticulum. A simple method to study the possible effects of nuclear envelope proteins on endoplasmic reticulum organisation is to analyze nuclear envelope protein overexpression. Here, we demonstrate that Lap2β overexpression can induce the formation of cytoplasmic vesicular structures derived from endoplasmic reticulum membranes. Correlative light and electron microscopy demonstrated that these vesicular structures were composed of a series of closely apposed membranes that were frequently arranged in a circular fashion. Although stacked endoplasmic reticulum cisternae were highly ordered, Lap2β could readily diffuse into and out of these structures into the surrounding reticulum. It appears that low-affinity interactions between cytoplasmic domains of Lap2β can reorganise reticular endoplasmic reticulum into stacked cisternae. Although the effect of one protein may be insignificant at low concentrations, the cumulative effect of many non-specialised proteins may be significant.
Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms
Römisch, Karin; Collie, Nicola; Soto, Nelyn; Logue, James; Lindsay, Margaret; Scheper, Wiep; Cheng, Chi-Hing C.
Secretory proteins enter the secretory pathway by translocation across the membrane of the endoplasmic reticulum (ER) via a channel formed primarily by the Sec61 protein. Protein translocation is highly temperature dependent in mesophilic organisms. We asked whether the protein translocation
Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs
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Winther, Anne-Marie Lund; Liu, Huizhen; Sonntag, Yonathan
fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate...
Are there different water requirements in different steps of a catalytic cycle? Hydration effects at the E1 and E2 conformers of sarcoplasmic reticulum Ca(2+)-ATPase studied in organic solvents with low amounts of water.
Barrabin, H; Scofano, H M; de Gómez-Puyou, M T; Gómez-Puyou, A
The Ca(2+)-ATPase from sarcoplasmic reticulum was transferred in an active form to a low-water system composed of toluene, phospholipids, and Triton X-100 (TPT). The Ca(2+)-ATPase activity in the TPT system with 4.0% water (by vol. was about 50% of the activity observed in all-aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme-catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.
Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion
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Ueda, Masashi; Li, Shimo; Itoh, Masanori; Wang, Miao-xing; Hayakawa, Miki; Islam, Saiful; Tana; Nakagawa, Kiyomi [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Chen, Huayue [Department of Anatomy, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Nakagawa, Toshiyuki, E-mail: email@example.com [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan)
The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.
Lipid homeostasis is involved in plasma membrane and endoplasmic reticulum stress in Pichia pastoris.
Zhang, Meng; Yu, Qilin; Liang, Chen; Zhang, Biao; Li, Mingchun
Maintaining cellular lipid composition is essential for many cell processes. Our previous study has demonstrated that Spt23 is an important transcription factor within the cell and responsible for the regulation of fatty acid desaturase genes. Disruption of SPT23 results in increased lipid saturation. In the present study, we found that lipid saturation caused by SPT23 deletion exhibited a growth defect under ethanol stress and increased chitin contents. Ergosterol synthesis-related genes were up-regulated to protect cells from plasma membrane damage in the presence of ethanol. The cell wall stress caused by increased chitin contents could not be attenuated by up-regulation of phospholipids synthesis-related genes in spt23Δ. Besides, lipid saturation induced expression of unfolded protein response (UPR) genes and reactive oxygen species (ROS) accumulation followed by activation of the cellular antioxidant system, which is associated with endoplasmic reticulum functions. Taken together, our data suggested that lipid homeostasis has a close connection with cell responses to both plasma membrane stress and endoplasmic reticulum stress. Copyright © 2016 Elsevier Inc. All rights reserved.
Myosin Va and Endoplasmic Reticulum Calcium Channel Complex Regulates Membrane Export during Axon Guidance
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Fumitaka Wada
Full Text Available During axon guidance, growth cones navigate toward attractive cues by inserting new membrane on the cue side. This process depends on Ca2+ release from endoplasmic reticulum (ER Ca2+ channels, but the Ca2+ sensor and effector governing this asymmetric vesicle export remain unknown. We identified a protein complex that controls asymmetric ER Ca2+-dependent membrane vesicle export. The Ca2+-dependent motor protein myosin Va (MyoVa tethers membrane vesicles to the ER via a common binding site on the two major ER Ca2+ channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, micromolar Ca2+ from ER channels triggers MyoVa-channel dissociation and the movement of freed vesicles to the cue side, enabling growth cone turning. MyoVa-Ca2+ channel interactions are required for proper long-range axon growth in developing spinal cord in vivo. These findings reveal a peri-ER membrane export pathway for Ca2+-dependent attraction in axon guidance.
The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria
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Fujimoto, Michiko [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Hayashi, Teruo, E-mail: firstname.lastname@example.com [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Su, Tsung-Ping, E-mail: email@example.com [Cellular Pathobiology Section, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States)
Highlights: Black-Right-Pointing-Pointer The endoplasmic reticulum subdomain termed MAM associates with mitochondria. Black-Right-Pointing-Pointer The biophysical role of lipids in the MAM-mitochondria association is unknown. Black-Right-Pointing-Pointer The in vitro membrane association assay was used to examine the role of lipids. Black-Right-Pointing-Pointer Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP{sub 3} receptor-mediated Ca{sup 2+} influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-{beta}-cyclodextrin (M{beta}C) significantly increases the association between MAMs and mitochondria, whereas M{beta}C saturated with cholesterol does not change the association. {sup 14}C-Serine pulse-labeling demonstrated that the treatment of living cells with M{beta}C decreases the level of de novo synthesized {sup 14}C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of
A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria
Tavassoli, Shabnam; Wong, Andrew K. O.; Choudhary, Vineet; Young, Barry P.; Loewen, Christopher J. R.; Prinz, William A.
Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth. PMID:25313861
Where the endoplasmic reticulum and the mitochondrion tie the knot: the mitochondria-associated membrane (MAM).
Raturi, Arun; Simmen, Thomas
More than a billion years ago, bacterial precursors of mitochondria became endosymbionts in what we call eukaryotic cells today. The true significance of the word "endosymbiont" has only become clear to cell biologists with the discovery that the endoplasmic reticulum (ER) superorganelle dedicates a special domain for the metabolic interaction with mitochondria. This domain, identified in all eukaryotic cell systems from yeast to man and called the mitochondria-associated membrane (MAM), has a distinct proteome, specific tethers on the cytosolic face and regulatory proteins in the ER lumen of the ER. The MAM has distinct biochemical properties and appears as ER tubules closely apposed to mitochondria on electron micrographs. The functions of the MAM range from lipid metabolism and calcium signaling to inflammasome formation. Consistent with these functions, the MAM is enriched in lipid metabolism enzymes and calcium handling proteins. During cellular stress situations, like an altered cellular redox state, the MAM alters its set of regulatory proteins and thus alters MAM functions. Notably, this set prominently comprises ER chaperones and oxidoreductases that connect protein synthesis and folding inside the ER to mitochondrial metabolism. Moreover, ER membranes associated with mitochondria also accommodate parts of the machinery that determines mitochondrial membrane dynamics and connect mitochondria to the cytoskeleton. Together, these exciting findings demonstrate that the physiological interactions between the ER and mitochondria are so bilateral that we are tempted to compare their relationship to the one of a married couple: distinct, but inseparable and certainly dependent on each other. In this paradigm, the MAM stands for the intracellular location where the two organelles tie the knot. Resembling "real life", the happy marriage between the two organelles prevents the onset of diseases that are characterized by disrupted metabolism and decreased lifespan
Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.
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Kyeong-Nam Yu
Full Text Available Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o- were exposed to 50 and 100 μg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA, mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.
Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control
Han, Sumin; Lone, Museer A.; Schneiter, Roger; CHANG, Amy
Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regul...
African swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum.
Andrés, G; García-Escudero, R; Simón-Mateo, C; Viñuela, E
During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.
Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction
Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.
Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994
Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum.
Raeymaekers, L; Wuytack, F; Eggermont, J; De Schutter, G; Casteels, R
1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates. PMID:6860302
Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.
Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J; Baluska, Frantisek
Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
Cloning of sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) from Caribbean spiny lobster Panulirus argus.
Mandal, A; Arunachalam, S C; Meleshkevitch, E A; Mandal, P K; Boudko, D Y; Ahearn, G A
We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using (45)Ca(2+) coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca(2+)-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.
Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.
Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer
Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.
Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex
Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.
Endoplasmic Reticulum-Mitochondria Communication Through Ca2+Signaling: The Importance of Mitochondria-Associated Membranes (MAMs).
Marchi, Saverio; Bittremieux, Mart; Missiroli, Sonia; Morganti, Claudia; Patergnani, Simone; Sbano, Luigi; Rimessi, Alessandro; Kerkhofs, Martijn; Parys, Jan B; Bultynck, Geert; Giorgi, Carlotta; Pinton, Paolo
The execution of proper Ca 2+ signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca 2+ released from the ER is "quasi-synaptically" transferred to mitochondrial matrix, where Ca 2+ stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca 2+ transfer is excessive and sustained, mitochondrial Ca 2+ overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca 2+ transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca 2+ flux and describe the activities of the various Ca 2+ players based on their primary intra-organelle localization.
Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol.
Infante, Rodney Elwood; Radhakrishnan, Arun
Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis.
Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates
Teuling, Eva; Ahmed, Suaad; Haasdijk, Elize; Demmers, Jeroen; Steinmetz, Michel O; Akhmanova, Anna; Jaarsma, Dick; Hoogenraad, Casper C
The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation
Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control
Han, Sumin; Lone, Museer A.; Schneiter, Roger; Chang, Amy
Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regulating sphingolipid synthesis. Phospholipid homeostasis is also affected in orm1Δ orm2Δ cells: the cells are inositol auxotrophs with impaired transcriptional regulation of genes encoding phospholipid biosynthesis enzymes. Strikingly, impaired growth of orm1Δ orm2Δ cells is associated with constitutive unfolded protein response, sensitivity to stress, and slow ER-to-Golgi transport. Inhibition of sphingolipid synthesis suppresses orm1Δ orm2Δ phenotypes, including ER stress, suggesting that disrupted sphingolipid homeostasis accounts for pleiotropic phenotypes. Thus, the yeast Orm proteins control membrane biogenesis by coordinating lipid homeostasis with protein quality control. PMID:20212121
Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells
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Mitsushi J. Ikemoto
Full Text Available Addicsin (Arl6ip5 is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1 and participates in the regulation of intracellular glutathione (GSH content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ, a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABAA receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na+/K+ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABAA receptor antagonist, picrotoxin, but not by a competitive GABAA receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.
A Chaperone Cascade Sorts Proteins for Posttranslational Membrane Insertion into the Endoplasmic Reticulum
National Research Council Canada - National Science Library
Wang, Fei; Brown, Emily C; Mak, Gary; Zhuang, Jimmy; Denic, Vladimir
... bilayer ( Rapoport, 2007 ). Cotranslational insertion by the SRP pathway, however, is not an option for membrane proteins that have their ER-targeting information contained in a single transmembrane domain (TMD) near the C terminus ( Kutay et al., 1995; Yabal et al., 2003 ). TMDs of these tail-anchored (TA) proteins emerge out of the rib...
Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis.
Priori, Silvia G; Chen, S R Wayne
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease occurring in patients with a structurally normal heart: the disease is characterized by life-threatening arrhythmias elicited by stress and emotion. In 2001, the ryanodine receptor was identified as the gene that is linked to CPVT; shortly thereafter, cardiac calsequestrin was implicated in the recessive form of the same disease. It became clear that abnormalities in intracellular Ca(2+) regulation could profoundly disrupt the electrophysiological properties of the heart. In this article, we discuss the molecular basis of the disease and the pathophysiological mechanisms that are impacting clinical diagnosis and management of affected individuals. As of today, the interaction between basic scientists and clinicians to understand CPVT and identify new therapeutic strategies is one of the most compelling examples of the importance of translational research in cardiology.
Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training
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Ørtenblad, Niels; Lunde, Per; Levin, Kasper
-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P RyR. No significant differences in Ca(2+) uptake rate and Ca(2+)-ATPase capacity were...
The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.
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Andree Hubber
Full Text Available The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P-binding domain first described in the effector DrrA (SidM. This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV, and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM and the endoplasmic reticulum (ER modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.
Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.
Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Leivar, Pablo; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles; Campos, Narciso
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells. © 2015 American Society of Plant Biologists. All Rights Reserved.
Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.
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Kelkar Anshuman
Full Text Available Abstract Background Protein translocation across the membrane of the Endoplasmic Reticulum (ER is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized. Results In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect. Conclusion Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.
Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and Golgi apparatus with (/sup 3/H)leucine
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Kappler, R.; Kristen, U.; Morre, D.J.
Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture,was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of (/sup 3/H)leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.
Analysis of Conditional Paralytic Mutants in Drosophila Sarco-Endoplasmic Reticulum Calcium ATPase Reveals Novel Mechanisms for Regulating Membrane Excitability
Sanyal, S.; Consoulas, C.; Kuromi, H.; Basole, A.; Mukai, L.; Kidokoro, Y.; Krishnan, K. S.; Ramaswami, M.
Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a gene...
Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes
Carette, J.E.; Lent, van J.; MacFarlance, S.A.; Wellink, J.E.; Kammen, van A.
Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have
A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.
Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali
Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.
Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells.
Fujii, Mizuki; Yoneda, Akihiro; Takei, Norio; Sakai-Sawada, Kaori; Kosaka, Marina; Minomi, Kenjiro; Yokoyama, Atsuro; Tamura, Yasuaki
Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Ultrastructure of Reissner's membrane in the rabbit
DEFF Research Database (Denmark)
Qvortrup, K.; Rostgaard, Jørgen; Bretlau, P.
Anatomy, Reissner's membrane, electron microscopy, tubulocisternal endoplasmic reticulum, subsurface cisterns, rabbit......Anatomy, Reissner's membrane, electron microscopy, tubulocisternal endoplasmic reticulum, subsurface cisterns, rabbit...
ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane
Joyce, Charles W.; Shelness, Gregory S.; Davis, Matthew A.; Lee, Richard G.; Skinner, Kelly; Anderson, Richard A.; Rudel, Lawrence L.
A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determi...
ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane
Joyce, Charles W.; Shelness, Gregory S.; Davis, Matthew A.; Lee, Richard G.; Skinner, Kelly; Anderson, Richard A.; Rudel, Lawrence L.
A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly. PMID:11071899
Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins.
Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe
Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.
EMC10 (Endoplasmic Reticulum Membrane Protein Complex Subunit 10) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial Infarction.
Reboll, Marc R; Korf-Klingebiel, Mortimer; Klede, Stefanie; Polten, Felix; Brinkmann, Eva; Reimann, Ines; Schönfeld, Hans-Joachim; Bobadilla, Maria; Faix, Jan; Kensah, George; Gruh, Ina; Klintschar, Michael; Gaestel, Matthias; Niessen, Hans W; Pich, Andreas; Bauersachs, Johann; Gogos, Joseph A; Wang, Yong; Wollert, Kai C
Clinical trials of bone marrow cell-based therapies after acute myocardial infarction (MI) have produced mostly neutral results. Treatment with specific bone marrow cell-derived secreted proteins may provide an alternative biological approach to improving tissue repair and heart function after MI. We recently performed a bioinformatic secretome analysis in bone marrow cells from patients with acute MI and discovered a poorly characterized secreted protein, EMC10 (endoplasmic reticulum membrane protein complex subunit 10), showing activity in an angiogenic screen. We investigated the angiogenic potential of EMC10 and its mouse homolog (Emc10) in cultured endothelial cells and infarcted heart explants. We defined the cellular sources and function of Emc10 after MI using wild-type, Emc10 -deficient, and Emc10 bone marrow-chimeric mice subjected to transient coronary artery ligation. Furthermore, we explored the therapeutic potential of recombinant Emc10 delivered by osmotic minipumps after MI in heart failure-prone FVB/N mice. Emc10 signaled through small GTPases, p21-activated kinase, and the p38 mitogen-activated protein kinase (MAPK)-MAPK-activated protein kinase 2 (MK2) pathway to promote actin polymerization and endothelial cell migration. Confirming the importance of these signaling events in the context of acute MI, Emc10 stimulated endothelial cell outgrowth from infarcted mouse heart explants via p38 MAPK-MK2. Emc10 protein abundance was increased in the infarcted region of the left ventricle and in the circulation of wild-type mice after MI. Emc10 expression was also increased in left ventricular tissue samples from patients with acute MI. Bone marrow-derived monocytes and macrophages were the predominant sources of Emc10 in the infarcted murine heart. Emc10 KO mice showed no cardiovascular phenotype at baseline. After MI, however, capillarization of the infarct border zone was impaired in KO mice, and the animals developed larger infarct scars and more
Protein transport into the human endoplasmic reticulum
Dudek, Johanna; Pfeffer, Stefan; Lee, Po-Hsien; Jung, Martin; Cavalié, Adolfo; Helms, Volkhard; Förster, Friedrich; Zimmermann, Richard
Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various
Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions
DEFF Research Database (Denmark)
Nielsen, J S; Sahlin, K; Ørtenblad, N
% and 16/100 Hz force ratio by 33%. The eSRCa2+ in fibres from non-stimulated muscles was 45 +/- 5% of the maximal loading capacity. After EC, eSRCa2+ per fibre CSA decreased by 38% (P = 0.05), and the maximal capacity of SR Ca2+ loading was depressed by 32%. There were no effects of EC on either...
Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle
DEFF Research Database (Denmark)
Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs
to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which...
Role of glycogen availability in sarcoplasmic reticulum Ca2+ kinetics in human skeletal muscle
DEFF Research Database (Denmark)
Ørtenblad, Niels; Nielsen, Joachim; Saltin, Bengt
Glucose is stored as glycogen in skeletal muscle. The importance of glycogen as a fuel during exercise has been recognized since the 1960s; however, little is known about the precise mechanism that relates skeletal muscle glycogen to muscle fatigue. We show that low muscle glycogen is associated...... with an impairment of muscle ability to release Ca(2+), which is an important signal in the muscle activation. Thus, depletion of glycogen during prolonged, exhausting exercise may contribute to muscle fatigue by causing decreased Ca(2+) release inside the muscle. These data provide indications of a signal...
Temperature dependence of cardiac sarcoplasmic reticulum and sarcolemma in the ventricle of catfish (Clarias gariepinus
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El-Sabry Abu-Amra
The sarcolemmal Ca2+ contribution of activator Ca2+ was greater at a test temperature of 30 °C as assessed by verapamil. Whereas the SR-Ca2+ contribution was higher at 20 and 30 °C and a frequency rate of 0.2 and 0.4 Hz as assessed by caffeine and adrenaline, respectively. Bradykinin potentiating factor (BPF7 which was isolated from jelly fish (Cassiopea andromeda decreased the cardiac force developed at a frequency rate of 0.2 Hz and a temperature of 20 °C, whereas it increased the force developed at frequency rates of 0.2 and 0.4 Hz at 30 °C. These results indicate that BPF7 may act like verapamil in reducing the cardiac force through blocking the sarcolemmal Ca2+ channels at low temperature and like adrenaline in an increase of the cardiac force developed at warm temperature and the high frequency rate through stimulation of SR-Ca2+ activator. Therefore, this study indicates that the sarcolemmal Ca2+ influx and the SR-Ca2+ release contributors of activator Ca2+ for cardiac force development in the catfish heart were significantly greater at warm temperature and at the pacing frequency rates of 0.2 and 0.4 Hz as assessed by verapamil, adrenaline, caffeine and BPF7. However, the relative contribution of the sarcolemmal Ca2+ influx in the development of cardiac force in the catfish heart was greater than that of SR-Ca2+ release.
The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins
DEFF Research Database (Denmark)
Madsen, Louise; Kriegenburg, Franziska; Lages Lino Vala, Andrea
The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolde...
Three-dimensional organization of a transcellular tubulocisternal endoplasmic reticulum in epithelial cells of Reissner's membrane in the guinea-pig
DEFF Research Database (Denmark)
Qvortrup, K; Rostgaard, Jørgen
The ultrastructure of the epithelial cells of Reissner's membrane (membrana vestibularis) in the guinea-pig is described following vascular perfusion with glutaraldehyde of live, anaesthetised and artificially respirated animals. Postfixation in a solution containing OsO4 and potassium ferricyanide...... is compared to other techniques used for preservation of Reissner's membrane. Each epithelial cell of Reissner's membrane is endowed with one kinocilium, one to four multivesicular bodies, and a number of intercalated bodies. The functional significance of the canalicular pathway is discussed....
Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1).
Desmond, Patrick F; Labuza, Amanda; Muriel, Joaquin; Markwardt, Michele L; Mancini, Allison E; Rizzo, Mark A; Bloch, Robert J
SERCA1, the sarco(endo)plasmic reticulum Ca(2+)-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca(2+) levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca(2+)-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca(2+)-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.
Wurie, Haja R; Buckett, Linda; Zammit, Victor A
Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1(-/-) mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.
The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins.
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Louise Madsen
Full Text Available The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.
Ractopamine-induced changes in sarcoplasmic proteome profile of ...
African Journals Online (AJOL)
Ractopamine is a beta-adrenergic agonist that increases leanness and carcass weight in finishing pigs. Our previous study observed that dietary ractopamine increased the abundance of several glycolytic enzymes in the sarcoplasmic proteome of post-rigor pork longissimus thoracis muscle. Pork semimembranosus is an ...
Endoplasmic Reticulum Stress and Obesity.
Yilmaz, Erkan
In recent years, the world has seen an alarming increase in obesity and closely associated with insulin resistance which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) play in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably other causes for obesity-related insulin resistance and inflammation. One of these appears to be endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.
Development of Endoplasmic Reticulum Stress during Experimental Oxalate Nephrolithiasis.
Motin, Yu G; Lepilov, A V; Bgatova, N P; Zharikov, A Yu; Motina, N V; Lapii, G A; Lushnikova, E L; Nepomnyashchikh, L M
Morphological and ultrastructural study of the kidney was performed in rats with oxalate nephrolithiasis. Specific features of endoplasmic reticulum stress were evaluated during nephrolithiasis and treatment with α-tocopherol. We observed the signs of endoplasmic reticulum stress with activation of proapoptotic pathways and injury to the cell lining in nephron tubules and collecting ducts. Ultrastructural changes were found in the organelles, nuclei, and cell membranes of epitheliocytes. A relationship was revealed between endoplasmic reticulum stress and oxidative damage, which developed at the early state of lithogenesis.
Expression of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes.
Corvazier, Elisabeth; Bredoux, Raymonde; Kovács, Tünde; Enouf, Jocelyne
The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.
A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.
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Pavel Montes de Oca Balderas
Full Text Available Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA receptor (NMDAR is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF labeling in rat cultured cortical astrocytes (rCCA of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(--2-amino-5-phosphonopentanoic acid (APV and Kynurenic acid (KYNA and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.
Bioactive electrospun fish sarcoplasmic proteins as a drug delivery system
DEFF Research Database (Denmark)
Stephansen, Karen; Chronakis, Ioannis S.; Jessen, Flemming
fiberswere insoluble in water. However, when exposed to proteolytic enzymes, the fibers were degraded. Thedegradation products of the FSP fibers proved to be inhibitors of the diabetes-related enzyme DPP-IV. TheFSP fibers may have biomedical applications, among others as a delivery system. To demonstrate......Nano-microfibers were made from cod (Gadus morhua) sarcoplasmic proteins (FSP) (Mwconcentration. Interestingly, the FSP...
Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites
Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J.; Baluska, Frantisek
Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum–plasma membrane (ER–PM) contact sites in leaf and root cells. The ER–PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER–PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER–PM contact sites. The VAP27-1-enriched ER–PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER–PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER–PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. PMID:27811083
Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.
Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie
Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.
Lipid Transport between the Endoplasmic Reticulum and Mitochondria
Flis, Vid V.
Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis. PMID:23732475
Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy
DEFF Research Database (Denmark)
Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc
Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium...
DEFF Research Database (Denmark)
Bach, Finn
Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...
A luminal flavoprotein in endoplasmic reticulum-associated degradation
DEFF Research Database (Denmark)
Riemer, Jan; Appenzeller-Herzog, Christian; Johansson, Linda
The quality control system of the endoplasmic reticulum (ER) discriminates between native and nonnative proteins. The latter are degraded by the ER-associated degradation (ERAD) pathway. Whereas many cytosolic and membrane components of this system are known, only few luminal players have been id...
Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis
Bravo-Sagua, R.; Rodriguez, A.E.; Kuzmicic, J.; Gutierrez, T.; Lopez-Crisosto, C.; Quiroga, C.; Díaz-Elizondo, J.; Chiong, M.; Gillette, T.G.; Rothermel, B.A.; Lavandero, S.
The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial–associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate. PMID:23228132
Solution-blown nanofiber mats from fish sarcoplasmic protein
DEFF Research Database (Denmark)
Sett, S.; Boutrup Stephansen, Karen; Yarin, A.L.
In the present work, solution-blowing was adopted to form nanofibers from fish sarcoplasmic proteins (FSPs). Nanofiber mats containing different weight ratios (up to 90/10) of FSP in the FSP/nylon 6 blended nanofibers were formed from formic acid solutions, and compared to electrospun fibers made...... techniques were similar, but with some exceptions. The fiber diameter of the electrospun fibers was slightly smaller than those made using solution-blowing, however in both cases the fiber diameter increased with increasing FSP content. Interestingly, for uniform fibers the stretchability of the fibers...... that the production rate of solution-blowing was increased 30-fold in relation to electrospinning. Overall, this study reveals FSP as an interesting biopolymeric alternative to synthetic polymers, and the introduction of FSP to nylon 6 provides a composite with controlled properties....
Endoplasmic Reticulum Malfunction in the Nervous System
Directory of Open Access Journals (Sweden)
Marek Michalak
Full Text Available Neurodegenerative diseases often have multifactorial causes and are progressive diseases. Some are inherited while others are acquired, and both vary greatly in onset and severity. Impaired endoplasmic reticulum (ER proteostasis, involving Ca2+ signaling, protein synthesis, processing, trafficking, and degradation, is now recognized as a key risk factor in the pathogenesis of neurological disorders. Lipidostasis involves lipid synthesis, quality control, membrane assembly as well as sequestration of excess lipids or degradation of damaged lipids. Proteostasis and lipidostasis are maintained by interconnected pathways within the cellular reticular network, which includes the ER and Ca2+ signaling. Importantly, lipidostasis is important in the maintenance of membranes and luminal environment that enable optimal protein processing. Accumulating evidence suggest that the loss of coordinate regulation of proteostasis and lipidostasis has a direct and negative impact on the health of the nervous system.
Voltage dependence of membrane charge movement and calcium release in frog skeletal muscle fibres.
Rakowski, R F; Best, P M; James-Kracke, M R
Voltage dependent membrane charge movement (gating current) and the release of Ca2+ from intracellular stores have been measured simultaneously in intact frog skeletal muscle fibres. Charge movement was measured using the three microelectrode voltage clamp technique. Ca2+ release was measured using the metallochromic indicator dye arsenazo III. Fibres were bathed in 2.3 X hypertonic solutions to prevent contraction. Rb+, tetraethylammonium and tetrodotoxin (TTX) were used to eliminate voltage-dependent ionic currents. The maximum rate of Ca2+ release from the sarcoplasmic reticulum in response to voltage-clamp step depolarizations to 0 mV was calculated using the dye-related parameters of model 2 of Baylor et al. (1983) and a method described in the Appendix for calculating a scaling factor (1 + p) that accounts for the additional Ca2+ buffering power of the indicator dye. The estimates of the maximum rate of Ca2+ release at 5-6 degrees C ranged from 3 to 19 microM ms-1 in the 17 fibres examined. The mean value was 8.9 +/- 1.1 microM ms-1 (S.E.M.) The maximum rate of Ca2+ release was linearly related to the magnitude of the nonlinear membrane change moved during suprathreshold depolarizing steps. The voltage dependence of charge movement and the maximum rate of Ca2+ releases were nearly identical at 6 degrees C. The voltage-dependence of the delay between the test step and the onset of Ca2+ release could be adequately described by an equation having the same functional form as the voltage dependence of nonlinear charge movement. The relationship between the test pulse voltage and the delay was shifted to more negative voltages and to shorter delays as the temperature was raised from 6 degrees C to 15 degrees C. The inactivation of Ca2+ release was found to occur at more negative holding voltages and to be more steeply voltage dependent than the immobilization of nonlinear membrane charge movement. The above data are discussed using the 'hypothetical coupler' model
Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling
Directory of Open Access Journals (Sweden)
Allyson eKranstuber
Full Text Available Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGE on long-lived proteins, including SR Ca2+ regulatory proteins. However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with Alagebrium Chloride (ALT-711 for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (by 27 and 41% vs. untreated diabetic rats, respectively, P<0.05. In cardiac myocytes, diabetes induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P<0.05; these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P<0.05, but preserved in the treated diabetic group compared to controls. Collectively, our result suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.
Fluorescent localization of membrane sites in glycerinated chicken skeletal muscle fibers and the relationship of these sites to the protein composition of the Z disc
Lazarides, Elias; Granger, Bruce L.
Didansyl derivatives of amino acids and N-phenyl-1-naphthylamine were used to localize membrane hydrophobic sites in glycerol-extracted chicken skeletal muscle fibers. Epifluorescence microscopy revealed that such sites coincide with the distribution of mitochondria, the transverse tubular (T) system and the sarcoplasmic reticulum (SR). They are specifically associated with myofibril Z lines and occasionally extend from one Z plane to the next longitudinally along the muscle fiber. The hydrophobic probes interact noncovalently with the Z lines, and their induced fluorescence can be eliminated by exposure of the myofibrils to ionic detergents, nonionic detergents, or phospholipase C, before or after addition of the hydrophobic label. Extraction of glycerinated fibers with 0.6 M KI removes the majority of sarcomeric actin and myosin and leaves a scaffold of longitudinally interconnected Z planes. Membrane fluorescence remains tightly associated with these Z planes and with the remnant mitochondria. Shearing of such scaffolds results in the cleavage of the longitudinal connections and the production of large sheets of interconnected, close-packed Z discs in a honeycomb-like array. Comparison of the localization of two Z disc proteins, desmin and α-actinin, with that of the membrane material reveals that α-actinin is localized in the interior of each myofibril Z disc whereas both desmin and the membrane material surround each disc. Thus, glycerination and KI extraction of muscle fibers leaves remnants of T system and SR membranes tightly associated with the Z disc honeycomb lattice. Because the Z discs are connected at their peripheries through the T system appear to the plasma membrane, desmin and this membrane structure appear to be connected throughout the whole Z plane up to and including the plasma membrane. The congruent localization of desmin and the T system strongly suggests that this molecule mediates the adhesion of this membrane system around each Z disc
Endoplasmic Reticulum (ER) Stress and Endocrine Disorders.
Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro
The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.
Endoplasmic Reticulum Stress and Type 2 Diabetes
Back, Sung Hoon; Kaufman, Randal J.
Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death. PMID:22443930
Endoplasmic Reticulum (ER Stress and Endocrine Disorders
Directory of Open Access Journals (Sweden)
Daisuke Ariyasu
Full Text Available The endoplasmic reticulum (ER is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR, which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI, Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2 are discussed in this article.
Junbo Hou; Min Yang
Lithium ion batteries have proven themselves the main choice of power sources for portable electronics. Besides consumer electronics, lithium ion batteries are also growing in popularity for military, electric vehicle, and aerospace applications. The present review attempts to summarize the knowledge about some selected membranes in lithium ion batteries. Based on the type of electrolyte used, literature concerning ceramic-glass and polymer solid ion conductors, microporous filter type separa...
Autophagosomal membranes assemble at ER-plasma membrane contact sites.
Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne
The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.
Endoplasmic reticulum-associated degradation of glycoproteins in plants
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Silvia eHüttner
Full Text Available In all eukaryotes the endoplasmic reticulum (ER has a central role in protein folding and maturation of secretory and membrane proteins. Upon translocation into the ER polypeptides are immediately subjected to folding and modifications involving the formation of disulfide bridges, assembly of subunits to multi-protein complexes and glycosylation. During these processes incompletely folded, terminally misfolded and unassembled proteins can accumulate which endanger the cellular homeostasis and subsequently the survival of cells and tissues. Consequently, organisms have developed a quality control system to cope with this problem and remove the unwanted protein load from the ER by a process collectively referred to as endoplasmic reticulum-associated degradation (ERAD pathway. Recent studies in Arabidopsis have identified plant ERAD components involved in the degradation of aberrant proteins and evidence was provided for a specific role in abiotic stress tolerance. In this short review we discuss our current knowledge about this important cellular pathway.
Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads.
Barrientos, G; Hidalgo, C
Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.
Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing
Capillary electrophoresis (CE) and reversed-phase high performance liquid chromatography (RP-HPLC) analysis were utilized to detect differences in the sarcoplasmic protein profiles of beef strip loins subjected to aging and hydrodynamic pressure processing (HDP) treatments. At 48 h postmortem, stri...
Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt.
Jo, Fusakazu; Jo, Hiromi; Hilzendeger, Aline M; Thompson, Anthony P; Cassell, Martin D; Rutkowski, D Thomas; Davisson, Robin L; Grobe, Justin L; Sigmund, Curt D
Endoplasmic reticulum stress has become an important mechanism in hypertension. We examined the role of endoplasmic reticulum stress in mediating the increased saline-intake and hypertensive effects in response to deoxycorticosterone acetate (DOCA)-salt. Intracerebroventricular delivery of the endoplasmic reticulum stress-reducing chemical chaperone tauroursodeoxycholic acid did not affect the magnitude of hypertension, but markedly decreased saline-intake in response to DOCA-salt. Increased saline-intake returned after tauroursodeoxycholic acid was terminated. Decreased saline-intake was also observed after intracerebroventricular infusion of 4-phenylbutyrate, another chemical chaperone. Immunoreactivity to CCAAT homologous binding protein, a marker of irremediable endoplasmic reticulum stress, was increased in the subfornical organ and supraoptic nucleus of DOCA-salt mice, but the signal was absent in control and CCAAT homologous binding protein-deficient mice. Electron microscopy revealed abnormalities in endoplasmic reticulum structure (decrease in membrane length, swollen membranes, and decreased ribosome numbers) in the subfornical organ consistent with endoplasmic reticulum stress. Subfornical organ-targeted adenoviral delivery of GRP78, a resident endoplasmic reticulum chaperone, decreased DOCA-salt-induced saline-intake. The increase in saline-intake in response to DOCA-salt was blunted in CCAAT homologous binding protein-deficient mice, but these mice exhibited a normal hypertensive response. We conclude that (1) brain endoplasmic reticulum stress mediates the saline-intake, but not blood pressure response to DOCA-salt, (2) DOCA-salt causes endoplasmic reticulum stress in the subfornical organ, which when attenuated by GRP78 blunts saline-intake, and (3) CCAAT homologous binding protein may play a functional role in DOCA-salt-induced saline-intake. The results suggest a mechanistic distinction between the importance of endoplasmic reticulum stress in
Cell death induced by endoplasmic reticulum stress
National Research Council Canada - National Science Library
Iurlaro, Raffaella; Muñoz‐Pinedo, Cristina
.... Many conditions that impose stress on cells, including hypoxia, starvation, infections and changes in secretory needs, challenge the folding capacity of the cell and promote endoplasmic reticulum stress...
Plant transducers of the endoplasmic reticulum unfolded protein response
Iwata, Yuji
The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.
Endoplasmic Reticulum Stress and Cancer
Yadav, Raj Kumar; Chae, Soo-Wan; Kim, Hyung-Ryong; Chae, Han Jung
The endoplasmic reticulum (ER) is the principal organelle responsible for multiple cellular functions including protein folding and maturation and the maintenance of cellular homeostasis. ER stress is activated by a variety of factors and triggers the unfolded protein response (UPR), which restores homeostasis or activates cell death. Multiple studies have clarified the link between ER stress and cancer, and particularly the involvement of the UPR. The UPR seems to adjust the paradoxical microenvironment of cancer and, as such, is one of resistance mechanisms against cancer therapy. This review describes the activity of different UPRs involved in tumorigenesis and resistance to cancer therapy. PMID:25337575
Membrane phospholipid fatty acid composition regulates cardiac SERCA activity in a hibernator, the Syrian hamster (Mesocricetus auratus.
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Sylvain Giroud
Full Text Available Polyunsaturated fatty acids (PUFA have strong effects on hibernation and daily torpor. Increased dietary uptake of PUFA of the n-6 class, particularly of Linoleic acid (LA, C18:2 n-6 lengthens torpor bout duration and enables animals to reach lower body temperatures (T(b and metabolic rates. As previously hypothesized, this well-known influence of PUFA may be mediated via effects of the membrane fatty acid composition on sarcoplasmic reticulum (SR Ca(2+-ATPase 2a (SERCA in the heart of hibernators. We tested the hypotheses that high proportions of n-6 PUFA in general, or specifically high proportions of LA (C18:2 n-6 in SR phospholipids (PL should be associated with increased cardiac SERCA activity, and should allow animals to reach lower minimum T(b in torpor. We measured activity of SERCA from hearts of hibernating and non-hibernating Syrian hamsters (Mesocricetus auratus in vitro at 35 °C. Further, we determined the PL fatty acid composition of the SR membrane of these hearts. We found that SERCA activity strongly increased as the proportion of LA in SR PL increased but was negatively affected by the content of Docosahexaenoic acid (DHA; C22:6 n-3. SR PL from hibernating hamsters were characterized by high proportions of LA and low proportions of DHA. As a result, SERCA activity was significantly higher during entrance into torpor and in torpor compared to inter-bout arousal. Also, animals with increased SERCA activity reached lower T(b during torpor. Interestingly, a subgroup of hamsters which never entered torpor but remained euthermic throughout winter displayed a phenotype similar to animals in summer. This was characterized by lower proportions of LA and increased proportions of DHA in SR membranes, which is apparently incompatible with torpor. We conclude that the PUFA composition of SR membranes affects cardiac function via modulating SERCA activity, and hence determines the minimum T(b tolerated by hibernators.
van Meer, G.
The thin membrane of the endoplasmic reticulum matures into the thick plasma membrane in the Golgi apparatus. Along the way, the concentrations of cholesterol and sphingolipids increase. Here, Gerrit van Meer discusses how this phenomenon may reflect an intricate lipid-protein sorting machinery.
Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres
Hernández-Ochoa, Erick O.; Schneider, Martin F.
Skeletal muscle excitation-contraction (E-C)1 coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)2 Ca2+ release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca2+, due to depolarization-initiated SR Ca2+ release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca2+ signalling properties of mouse skeletal muscle membranes are being investigated. PMID:22306655
Sarcoplasmic masses in the skeletal muscle of a stranded pigmy sperm whale (Kogia breviceps).
Sierra, Eva; de los Monteros, Antonio Espinosa; Fernández, Antonio; Arbelo, Manuel; Caballero, María José; Rivero, Miguel; Herráez, Pedro
We measured the abundance of sarcoplasmic masses within skeletal muscle myocytes of an adult female stranded pigmy sperm whale (Kogia breviceps). The presence of these masses in other species has been reported in association with myopathies, including myotonic dystrophy, the most frequently related pathology. Other histopathologic muscle changes included a high number of internal nuclei, variations in fiber size and shape, and the predominance of type I fibers.
Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?
Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R
A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.
Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.
Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J
Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.
Endoplasmic reticulum stress in lung disease
Directory of Open Access Journals (Sweden)
Stefan J. Marciniak
Full Text Available Exposure to inhaled pollutants, including fine particulates and cigarette smoke is a major cause of lung disease in Europe. While it is established that inhaled pollutants have devastating effects on the genome, it is now recognised that additional effects on protein folding also drive the development of lung disease. Protein misfolding in the endoplasmic reticulum affects the pathogenesis of many diseases, ranging from pulmonary fibrosis to cancer. It is therefore important to understand how cells respond to endoplasmic reticulum stress and how this affects pulmonary tissues in disease. These insights may offer opportunities to manipulate such endoplasmic reticulum stress pathways and thereby cure lung disease.
Integrated endoplasmic reticulum stress responses in cancer
National Research Council Canada - National Science Library
Moenner, Michel; Pluquet, Olivier; Bouchecareilh, Marion; Chevet, Eric
The endoplasmic reticulum (ER) has emerged as a major site of cellular homeostasis regulation, particularly in the unfolded protein response, which is being found to play a major role in cancer and many other diseases...
Molecular cloning and functional expression of a novel human gene encoding two 41-43 kDa skeletal muscle internal membrane proteins.
Bouju, S; Lignon, M F; Piétu, G; Le Cunff, M; Léger, J J; Auffray, C; Dechesne, C A
Systematic analysis of gene transcript repertoires prepared from libraries made with various specific human tissues permitted isolation of many partially sequenced cDNA clones. A few of these represented novel genes with limited or no similarity to known genes from humans or other species. The present study set out to isolate and sequence the full-length cDNA corresponding to one of these novel human transcripts, and identify the corresponding protein product at the subcellular level. Current sequence analyses have revealed that the protein contains a hydrophobic N-terminal segment and an internal leucine-zipper motif. Numerous sites of putative post-translational modifications, such as N-linked glycosylation, myristoylation and phosphorylation sites, were also identified. Using one monoclonal antibody raised against a recombinant fragment, two different 41-43 kDa proteins were detected in human skeletal muscle, heart and placenta homogenates at various ratios. Both immunodetected protein products of the novel human gene were distributed in the transverse tubules and/or near the junctional sarcoplasmic reticulum within skeletal muscle cells. Both proteins had physical properties believed to be attributable to integral membrane components. Finally, the GENX-3414 gene was chromosomally localized at position 4q24-q25.
Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum.
Zito, Ester; Buono, Mario; Pepe, Stefano; Settembre, Carmine; Annunziata, Ida; Surace, Enrico Maria; Dierks, Thomas; Monti, Maria; Cozzolino, Marianna; Pucci, Piero; Ballabio, Andrea; Cosma, Maria Pia
Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.
Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad.
Launikonis, B S; Stephenson, D G
1. Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation-contraction (E-C) coupling. The cholesterol content of membranes was manipulated with methyl-beta-cyclodextrin (MbetaCD). 2. In mechanically skinned fibres, depletion of membrane cholesterol with MbetaCD caused a dose- and time-dependent decrease in transverse tubular (t)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mM MbetaCD but were not affected after 2 min in the presence of a 10 mM MbetaCD-1 mM cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MbetaCD : cholesterol ratio at 10 mM MbetaCD, indicating that the inhibitory effect of MbetaCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3-4 h in the presence of 10 mM MbetaCD. 3. The duration of TSDIFRs increased markedly soon (laser scanning microscopy revealed that the integrity of the t-system was not compromised by either intra- or extracellular application of 10 mM MbetaCD and that a large [Ca(2+)] gradient was maintained across the t-system. 5. Membrane cholesterol depletion caused rapid depolarization of the polarized t-system as shown independently by spontaneous TSDIFRs induced by MbetaCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC(4)(3)) in the presence of MbetaCD. This rapid depolarization of the t-system by cholesterol depletion was not prevented by blocking the Na(+) channels with TTX (10 microM) or the L-type Ca(2+) channels with Co(2+) (5 mM). 6. The results demonstrate that cholesterol is important for maintaining the functional integrity of the t-system and sarcoplasmic reticulum, probably by having specific
Calcium pumps of plasma membrane and cell interior
DEFF Research Database (Denmark)
Strehler, Emanuel E; Treiman, Marek
/endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue...
Endoplasmic Reticulum Stress in Sepsis
Khan, Mohammad Moshahid; Yang, Weng-Lang; Wang, Ping
Sepsis is an enormous public health issue and the leading cause of death in critically ill patients in intensive care units (ICU). Overwhelming inflammation, characterized by cytokine storm, oxidative threats, and neutrophil sequestration is an underlying component of sepsis-associated organ failure. Despite recent advances in sepsis research, there is still no effective treatment available beyond the standard of care and supportive therapy. To reduce sepsis-related mortality, a better understanding of the biological mechanism associated with the sepsis is essential. Endoplasmic reticulum (ER), a subcellular organelle is responsible for the facilitation of protein folding and assembly and involved in several other physiological activities. Under the stress and inflammation condition, ER loses the homeostasis in its function, which is termed as ER stress. During ER stress, unfolded protein response (UPR) is activated to restore ER function to its normal balance. However, once the stress is beyond the compensatory capacity of UPR or protracted, the apoptosis would be initiated by triggering cell injuries, even to cell death. As such, ER stress and UPR are reported to be implicated in several pathological and inflammatory conditions. Although the detrimental role of ER stress during infections has been demonstrated, there is growing evidences that ER stress participate in the pathogenesis of sepsis. In this review, we summarize the current research in the context of ER stress and UPR signaling associated with sepsis and its related clinical conditions, such as trauma- hemorrhage, and ischemia/reperfusion (I/R) injury. We also discuss the potential implication of ER stress as a novel therapeutic target and prognostic marker in patients with sepsis. PMID:26125088
Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.
Missiroli, Sonia; Morganti, Claudia; Giorgi, Carlotta; Pinton, Paolo
Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes
DEFF Research Database (Denmark)
Boutrup Stephansen, Karen; Mattebjerg, Maria Ahlm; Wattjes, Jasper
Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged...... fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta...
Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.
Manfredi, Giovanni; Kawamata, Hibiki
Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS. Copyright © 2015 Elsevier Inc. All rights reserved.
Cell death induced by endoplasmic reticulum stress.
Iurlaro, Raffaella; Muñoz-Pinedo, Cristina
The endoplasmic reticulum is an organelle with multiple functions. The synthesis of transmembrane proteins and proteins that are to be secreted occurs in this organelle. Many conditions that impose stress on cells, including hypoxia, starvation, infections and changes in secretory needs, challenge the folding capacity of the cell and promote endoplasmic reticulum stress. The cellular response involves the activation of sensors that transduce signaling cascades with the aim of restoring homeostasis. This is known as the unfolded protein response, which also intersects with the integrated stress response that reduces protein synthesis through inactivation of the initiation factor eIF2α. Central to the unfolded protein response are the sensors PERK, IRE1 and ATF6, as well as other signaling nodes such as c-Jun N-terminal kinase 1 (JNK) and the downstream transcription factors XBP1, ATF4 and CHOP. These proteins aim to restore homeostasis, but they can also induce cell death, which has been shown to occur by necroptosis and, more commonly, through the regulation of Bcl-2 family proteins (Bim, Noxa and Puma) that leads to mitochondrial apoptosis. In addition, endoplasmic reticulum stress and proteotoxic stress have been shown to induce TRAIL receptors and activation of caspase-8. Endoplasmic reticulum stress is a common feature in the pathology of numerous diseases because it plays a role in neurodegeneration, stroke, cancer, metabolic diseases and inflammation. Understanding how cells react to endoplasmic reticulum stress can accelerate discovery of drugs against these diseases. © 2015 FEBS.
Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability.
Canto, Anna C V C S; Suman, Surendranath P; Nair, Mahesh N; Li, Shuting; Rentfrow, Gregg; Beach, Carol M; Silva, Teofilo J P; Wheeler, Tommy L; Shackelford, Steven D; Grayson, Adria; McKeith, Russell O; King, D Andy
The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color. Copyright © 2014 Elsevier Ltd. All rights reserved.
Endoplasmic Reticulum Stress-Related Inflammation and Cardiovascular Diseases
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Tomomi Gotoh
Full Text Available The endoplasmic reticulum (ER is the site of synthesis and maturation of proteins designed for secretion or for localization on the cell membrane. Various types of stress from both inside and outside cells disturb ER function, thus causing unfolded or misfolded proteins to accumulate in the ER. To improve and maintain the ER functions against such stresses, the ER stress response pathway is activated. However, when the stress is prolonged or severe, apoptosis pathways are activated to remove damaged cells. It was recently reported that the ER stress pathway is also involved in the inflammatory response, whereby inflammation induces ER stress, and ER stress induces an inflammatory response. Therefore, the ER stress response pathway is involved in various diseases, including cardiovascular diseases such as atherosclerosis and ischemic diseases, in various ways. The ER stress pathway may represent a novel target for the treatment of these diseases.
The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum.
Broadway, Neil M; Pease, Richard J.; Birdsey, Graeme; Shayeghi, Majid; Turner, Nigel A; David Saggerson, E
Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 54...
Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs
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Mahmmoud, Yasser Ahmed; Gaster, Michel
agents. EXPERIMENTAL APPROACH: Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. KEY RESULTS: Studies on SR vesicles showed that the endogenous...... lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C......-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin...
Angiotensin receptor antagonism and angiotensin converting enzyme inhibition improve diastolic dysfunction and Ca2+-ATPase expression in the sarcoplasmic reticulum in hypertensive cardiomyopathy
Flesch, M; Schiffer, F; Zolk, O; Pinto, Y; Stasch, JP; Knorr, A; Ettelbruck, S; Bohm, M
Background Hypertensive cardiomyopathy is a major risk factor for the development of chronic heart failure, Objective To investigate whether treatment with an angiotensin converting enzyme inhibitor (ACEI) or with an angiotensin type 1 receptor antagonist (AT(1)-RA) is sufficient to prevent the
Angiotensin receptor antagonism and angiotensin converting enzyme inhibition improve diastolic dysfunction and Ca(2+)-ATPase expression in the sarcoplasmic reticulum in hypertensive cardiomyopathy
Flesch, M.; Schiffer, F.; Zolk, O.; Pinto, Y.; Stasch, J. P.; Knorr, A.; Ettelbrück, S.; Böhm, M.
BACKGROUND: Hypertensive cardiomyopathy is a major risk factor for the development of chronic heart failure. OBJECTIVE: To investigate whether treatment with an angiotensin converting enzyme inhibitor (ACEI) or with an angiotensin type 1 receptor antagonist (AT1-RA) is sufficient to prevent the
Sarcoplasmic reticulum Ca2+ uptake rate and endogenous content in MHC I and MHC II fibres of human skeletal muscle following prolonged exercise in highly trained
DEFF Research Database (Denmark)
Ørtenblad, Niels; Nielsen, Jens Steen
load time was increased by 8% (P direct measures that the SR Ca2+ loading time decreases in MHCI fibres following prolonged cycling exercise in highly trained humans, however...... no differences in eSR content between the fibre types before exercise and no change with exhaustive exercise. The loading time was 17% slower in MHC II fibres (13.4 ± 0.2 vs 15.7 ± 0.2 sec, MHCI and MHCII respectively). However, the maximum loading capacity was higher in MHC II fibres. Following exercise the SR...
Connecting the cytoskeleton to the endoplasmic reticulum and Golgi.
Gurel, Pinar S; Hatch, Anna L; Higgs, Henry N
A tendency in cell biology is to divide and conquer. For example, decades of painstaking work have led to an understanding of endoplasmic reticulum (ER) and Golgi structure, dynamics, and transport. In parallel, cytoskeletal researchers have revealed a fantastic diversity of structure and cellular function in both actin and microtubules. Increasingly, these areas overlap, necessitating an understanding of both organelle and cytoskeletal biology. This review addresses connections between the actin/microtubule cytoskeletons and organelles in animal cells, focusing on three key areas: ER structure and function; ER-to-Golgi transport; and Golgi structure and function. Making these connections has been challenging for several reasons: the small sizes and dynamic characteristics of some components; the fact that organelle-specific cytoskeletal elements can easily be obscured by more abundant cytoskeletal structures; and the difficulties in imaging membranes and cytoskeleton simultaneously, especially at the ultrastructural level. One major concept is that the cytoskeleton is frequently used to generate force for membrane movement, with two potential consequences: translocation of the organelle, or deformation of the organelle membrane. While initially discussing issues common to metazoan cells in general, we subsequently highlight specific features of neurons, since these highly polarized cells present unique challenges for organellar distribution and dynamics. Copyright © 2014 Elsevier Ltd. All rights reserved.
GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum
DEFF Research Database (Denmark)
Marzec, Michal; Eletto, Davide; Argon, Yair
Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94...... shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme...
Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins
Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.
Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a
Arachidonoyl-specific diacylglycerol kinase ε and the endoplasmic reticulum
Directory of Open Access Journals (Sweden)
Tomoyuki Nakano
Full Text Available The endoplasmic reticulum (ER comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK that phosphorylates diacylglycerol (DG to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER.
Emerging role of the endoplasmic reticulum in peroxisome biogenesis
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Gaurav eAgrawal
Full Text Available During the past few years, we have witnessed a paradigm shift in our long-standing concept of peroxisome biogenesis. Recent biochemical and morphological studies have revealed a primary role of the endoplasmic reticulum (ER in the de novo formation of peroxisomes, thus challenging the prevalent model invoking growth and division of pre-existing peroxisomes. Importantly, a novel sorting process has been recently defined at the ER that segregates and assembles specific sets of peroxisomal membrane proteins into distinct pre-peroxisomal vesicular carriers (ppVs that later undergo heterotypic fusion to form mature peroxisomes. Consequently, the emerging model has redefined the function of many peroxins (most notably Pex3, Pex19 and Pex25 and assigned them novel roles in vesicular budding and subsequent peroxisome assembly. These advances establish a novel intracellular membrane trafficking route between the ER and peroxisomes, but the components remain elusive. This review will provide a historical perspective and focus on recent developments in the emerging role of the ER in peroxisome biogenesis.
Endoplasmic Reticulum Stress and Associated ROS
Zeeshan, Hafiz Maher Ali; Lee, Geum Hwa; Kim, Hyung-Ryong; Chae, Han-Jung
The endoplasmic reticulum (ER) is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS). Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI)-endoplasmic reticulum oxidoreductin (ERO)-1, glutathione (GSH)/glutathione disuphide (GSSG), NADPH oxidase 4 (Nox4), NADPH-P450 reductase (NPR), and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases. PMID:26950115
Endoplasmic Reticulum Stress and Associated ROS
Directory of Open Access Journals (Sweden)
Hafiz Maher Ali Zeeshan
Full Text Available The endoplasmic reticulum (ER is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS. Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI-endoplasmic reticulum oxidoreductin (ERO-1, glutathione (GSH/glutathione disuphide (GSSG, NADPH oxidase 4 (Nox4, NADPH-P450 reductase (NPR, and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.
Protein Secretion and the Endoplasmic Reticulum
Benham, Adam M.
In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisti...
Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury.
Sun, Guo-Zhu; Gao, Fen-Fei; Zhao, Zong-Mao; Sun, Hai; Xu, Wei; Wu, Li-Wei; He, Yong-Chang
Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endoplasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of fluid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These findings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.
Dihydropyridine-sensitive ion currents and charge movement in vesicles derived from frog skeletal muscle plasma membranes.
Camacho, J; Carapia, A; Calvo, J; García, M C; Sánchez, J A
1. Whole-cell voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the electrophysiological properties of dihydropyridine (DHP) receptors. This preparation allows control of the composition of the internal medium and the recording of currents, without the influence of the sarcoplasmic reticulum (SR). 2. In solutions containing Ba2+, Bay K 8644-sensitive, L-type inward currents were recorded. Peak Ba2+ currents (IBa) averaged 3.0 microA microF-1 and inactivated in a voltage-dependent manner. Half-maximal steady-state inactivation occurred at -40 mV. No major facilitation of tail currents was observed. 3. The time course of activation of L-type Ca2+ channels was voltage dependent and 10 times faster than that in muscle fibres; the current density values were also much lower. 4. Lowering [Mg2+]i from 2 to 0.1 mM shifted the time to peak of IBa versus voltage relation by -13 mV. 5. In solutions that contained mostly impermeant ions, non-linear capacitive currents were recorded. Charge movement with properties resembling charge 1 was observed in polarized vesicles. The charge movement depended on voltage with Boltzmann parameters: Qmax (maximum charge), 45.6 nC microF-1; V (potential at which Q = 0.5 Qmax), -58.4 mV; and k (slope factor), 22. 3 mV. There was no indication of the presence of Qgamma (the 'hump' component of charge movement). 6. In depolarized vesicles, non-linear currents were observed during hyperpolarizing pulses. The currents produced an excessive charge during 'on' transients only. Charge during 'off' transients was linear from -180 to +60 mV. There was no evidence of the presence of charge 2.
Flavour formation from hydrolysis of pork sarcoplasmic protein extract by a unique LAB culture isolated from Harbin dry sausage.
Chen, Qian; Liu, Qian; Sun, Qinxiu; Kong, Baohua; Xiong, Youling
The lactic acid bacteria Pediococcus pentosaceus, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum isolated from Harbin dry sausage were assessed for their protein hydrolysis and flavour development in pork muscle sarcoplasmic protein extracts. Gel electrophoresis indicated that sarcoplasmic proteins were degraded by all of the strains, especially by P. pentosaceus and L. curvatus. Trichloroacetic acid-soluble peptides increased in all of the samples (P < 0.05), especially samples inoculated with P. pentosaceus. Samples inoculated with P. pentosaceus and L. curvatus had higher free amino acid contents than did the other two strains(P < 0.05), and glutamic acid and alanine appeared to be the predominant free amino acids. The volatile compound analysis indicated that the highest aldehydes, alcohols and acid contents were found in the sample with P. pentosaceus followed by L. curvatus. The results revealed that P. pentosaceus could be appropriate for use as a meat starter culture.
Sex differences play a role in cardiac endoplasmic reticulum stress (ERS) and ERS-initiated apoptosis induced by pressure overload and thapsigargin.
Sari, Flori R; Watanabe, Kenichi; Widyantoro, Bambang; Thandavarayan, Rajarajan A; Harima, Meilei; Kodama, Makoto; Aizawa, Yoshifusa
Excessive endoplasmic reticulum stress (ERS) triggers myocardial apoptosis. Sex differences appear to be an important determinant in the occurrence of stress and apoptosis through many pathways, but the roles of sex differences in the cardiac ERS and ERS-initiated apoptosis are largely unknown. In the present study, we investigated the in vivo role of sex differences in the cardiac ERS and apoptosis elicited by ascending aortic banding surgery or thapsigargin (Thap) injection using male and female C57BL/6 JAX mice. The surgery significantly increased the expression levels of cardiac glucose-regulated protein (GRP)78 and CCAAT/enhancer binding protein homology protein (CHOP) protein, increased the myocardial apoptosis and decreased the sarcoplasmic reticulum Ca(2+)-ATPase isoform (SERCA)2 immunoreactivity in the male mice relative to female mice. Furthermore, during ERS induction using Thap, myocardial apoptosis and the expression levels of cardiac GRP78, inositol-requiring enzyme (Ire)1α and tumor necrosis factor receptor-associated factor (TRAF)2 were significantly increased in male mice relative to female mice. Sex differences significantly affected the above results. Our data suggest that sex differences affected the response of myocardial tissues in dealing with cardiac ERS and further result of ERS, apoptosis, at least in part through the regulation of SERCA2, CHOP, Ire1α and TRAF2. Copyright © 2011. Published by Elsevier Inc.
Ultrasonography of the reticulum in 30 healthy Saanen goats
Braun, Ueli; Jacquat, Désirée
Abstract Background The reticulum plays a crucial role in the ruminant digestive tract because the primary cycle of rumen motility always starts with a reticular contraction. In contrast to cattle, there are only few results on the ultrasonographic examination of the reticulum in goats. Therefore, it was the goal of the present study, to describe the results of ultrasonography of the reticulum of 30 healthy Saanen goats. Methods Ultrasonography was carried out on standing, non-sedated animals...
Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases
Liu, Mei-qing; Chen, Zhe; Chen, Lin-xi
Endoplasmic reticulum is a principal organelle responsible for folding, post-translational modifications and transport of secretory, luminal and membrane proteins, thus palys an important rale in maintaining cellular homeostasis. Endoplasmic reticulum stress (ERS) is a condition that is accelerated by accumulation of unfolded/misfolded proteins after endoplasmic reticulum environment disturbance, triggered by a variety of physiological and pathological factors, such as nutrient deprivation, altered glycosylation, calcium depletion, oxidative stress, DNA damage and energy disturbance, etc. ERS may initiate the unfolded protein response (UPR) to restore cellular homeostasis or lead to apoptosis. Numerous studies have clarified the link between ERS and cardiovascular diseases. This review focuses on ERS-associated molecular mechanisms that participate in physiological and pathophysiological processes of heart and blood vessels. In addition, a number of drugs that regulate ERS was introduced, which may be used to treat cardiovascular diseases. This review may open new avenues for studying the pathogenesis of cardiovascular diseases and discovering novel drugs targeting ERS. PMID:26838072
Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood-Brain Barrier Damage.
Qie, Xiaojuan; Wen, Di; Guo, Hongyan; Xu, Guanjie; Liu, Shuai; Shen, Qianchao; Liu, Yi; Zhang, Wenfang; Cong, Bin; Ma, Chunling
Methamphetamine (METH) abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood-brain barrier (BBB). Herein, we explored the potential mechanism of endoplasmic reticulum (ER) stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3) cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS) and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo) and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption.
Endoplasmic Reticulum Stress Mediates Methamphetamine-Induced Blood–Brain Barrier Damage
Directory of Open Access Journals (Sweden)
Xiaojuan Qie
Full Text Available Methamphetamine (METH abuse causes serious health problems worldwide, and long-term use of METH disrupts the blood–brain barrier (BBB. Herein, we explored the potential mechanism of endoplasmic reticulum (ER stress in METH-induced BBB endothelial cell damage in vitro and the therapeutic potential of endoplasmic reticulum stress inhibitors for METH-induced BBB disruption in C57BL/6J mice. Exposure of immortalized BMVEC (bEnd.3 cells to METH significantly decreased cell viability, induced apoptosis, and diminished the tightness of cell monolayers. METH activated ER stress sensor proteins, including PERK, ATF6, and IRE1, and upregulated the pro-apoptotic protein CHOP. The ER stress inhibitors significantly blocked the upregulation of CHOP. Knockdown of CHOP protected bEnd.3 cells from METH-induced cytotoxicity. Furthermore, METH elevated the production of reactive oxygen species (ROS and induced the dysfunction of mitochondrial characterized by a Bcl2/Bax ratio decrease, mitochondrial membrane potential collapse, and cytochrome c. ER stress release was partially reversed by ROS inhibition, and cytochrome c release was partially blocked by knockdown of CHOP. Finally, PBA significantly attenuated METH-induced sodium fluorescein (NaFluo and Evans Blue leakage, as well as tight junction protein loss, in C57BL/6J mice. These data suggest that BBB endothelial cell damage was caused by METH-induced endoplasmic reticulum stress, which further induced mitochondrial dysfunction, and that PBA was an effective treatment for METH-induced BBB disruption.
Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum
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S. Andrei Anghel
Full Text Available Members of the evolutionarily conserved Oxa1/Alb3/YidC family mediate membrane protein biogenesis at the mitochondrial inner membrane, chloroplast thylakoid membrane, and bacterial plasma membrane, respectively. Despite their broad phylogenetic distribution, no Oxa1/Alb3/YidC homologs are known to operate in eukaryotic cells outside the endosymbiotic organelles. Here, we present bioinformatic evidence that the tail-anchored protein insertion factor WRB/Get1, the "endoplasmic reticulum (ER membrane complex" subunit EMC3, and TMCO1 are ER-resident homologs of the Oxa1/Alb3/YidC family. Topology mapping and co-evolution-based modeling demonstrate that Get1, EMC3, and TMCO1 share a conserved Oxa1-like architecture. Biochemical analysis of human TMCO1, the only homolog not previously linked to membrane protein biogenesis, shows that it associates with the Sec translocon and ribosomes. These findings suggest a specific biochemical function for TMCO1 and define a superfamily of proteins—the "Oxa1 superfamily"—whose shared function is to facilitate membrane protein biogenesis.
A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress
Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d'Hellencourt, Christian; Ravanan, Palaniyandi
Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress. PMID:25120434
Endoplasmic reticulum stress in insulin resistance and diabetes.
Guerrero-Hernández, Agustin; Leon-Aparicio, Daniel; Chavez-Reyes, Jesus; Olivares-Reyes, Jesus A; DeJesus, Silvia
The endoplasmic reticulum is the main intracellular Ca(2+) store for Ca(2+) release during cell signaling. There are different strategies to avoid ER Ca(2+) depletion. Release channels utilize first Ca(2+)-bound to proteins and this minimizes the reduction of the free luminal [Ca(2+)]. However, if release channels stay open after exhaustion of Ca(2+)-bound to proteins, then the reduction of the free luminal ER [Ca(2+)] (via STIM proteins) activates Ca(2+) entry at the plasma membrane to restore the ER Ca(2+) load, which will work provided that SERCA pump is active. Nevertheless, there are several noxious conditions that result in decreased activity of the SERCA pump such as oxidative stress, inflammatory cytokines, and saturated fatty acids, among others. These conditions result in a deficient restoration of the ER [Ca(2+)] and lead to the ER stress response that should facilitate recovery of the ER. However, if the stressful condition persists then ER stress ends up triggering cell death and the ensuing degenerative process leads to diverse pathologies; particularly insulin resistance, diabetes and several of the complications associated with diabetes. This scenario suggests that limiting ER stress should decrease the incidence of diabetes and the mobility and mortality associated with this illness. Copyright © 2014 Elsevier Ltd. All rights reserved.
Endoplasmic Reticulum-Mediated Protein Quality Control in Arabidopsis
Directory of Open Access Journals (Sweden)
Jianming eLi
Full Text Available A correct three-dimensional structure is crucial for the physiological functions of a protein, yet the folding of proteins to acquire native conformation is a fundamentally error-prone process. Eukaryotic organisms have evolved a highly conserved endoplasmic reticulum-mediated protein quality control (ERQC mechanism to monitor folding processes of secretory and membrane proteins, allowing export of only correctly folded proteins to their physiological destinations, retaining incompletely/mis-folded ones in the ER for additional folding attempts, marking and removing terminally-misfolded ones via a unique multiple-step degradation process known as ER-associate degradation (ERAD. Most of our current knowledge on ERQC and ERAD came from genetic and biochemical investigations in yeast and mammalian cells. Recent studies in the reference plant Arabidopsis thaliana uncovered homologous components and similar mechanisms in plants for monitoring protein folding and for retaining, repairing, and removing misfolded proteins. These studies also revealed critical roles of the plant ERQC/ERAD systems in regulating important biochemical/physiological processes, such as abiotic stress tolerance and plant defense. In this review, we discuss our current understanding about the molecular components and biochemical mechanisms of the plant ERQC/ERAD system in comparison to yeast and mammalian systems.
Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases.
Rieusset, Jennifer
The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases. Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes (MAM), in order to exchange metabolites and calcium (Ca 2+ ) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca 2+ signaling in both healthy and pathological contexts. Then we focus on recent data highlighting MAM as important hubs for hormone and nutrient signaling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss how chronic ER-mitochondria miscommunication could participate to hepatic metabolic diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.
Coordination of Endoplasmic Reticulum (ER) Signaling During Maize Seed Development
Energy Technology Data Exchange (ETDEWEB)
Boston, Rebecca S.
Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesize storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies. Our underlying hypothesis is that the endoplasmic reticulum (ER) stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presence of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly. The capacity of the ER to deposit these aberrant proteins in protein bodies during a period that extends several weeks provides an excellent model for deconvoluting the ER stress response of plants. We have focused in this project on the means by which the ER senses and responds to functional perturbations and the underlying intracellular communication that occurs among biosynthetic, trafficking and degradative pathways for proteins during seed development.
The endoplasmic reticulum in plant immunity and cell death
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Patrick eSchäfer
Full Text Available The endoplasmic reticulum (ER is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programmed cell death (PCD in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signalling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants.
Stress responses from the endoplasmic reticulum in cancer
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Hironori eKato
Full Text Available The endoplasmic reticulum (ER is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR. The UPR also contributes to the regulation of various intracellular signalling pathways such as calcium signalling and lipid signalling. More recently, the mitochondria-associated ER membrane (MAM, which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signalling, inflammatory signalling, the autophagic response, and the UPR. Interestingly, in cancer, these signalling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signalling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM.
[From endoplasmic reticulum to Golgi apparatus: a secretory pathway controlled by signal molecules].
Wang, Jiasheng; Luo, Jianhong; Zhang, Xiaomin
Protein transport from endoplasmic reticulum (ER) to Golgi apparatus has long been known to be a central process for protein quality control and sorting. Recent studies have revealed that a large number of signal molecules are involved in regulation of membrane trafficking through ER, ER-Golgi intermediate compartment and Golgi apparatus. These molecules can significantly change the transport rate of proteins by regulating vesicle budding and fusion. Protein transport from ER to Golgi apparatus is not only controlled by signal pathways triggered from outside the cell, it is also regulated by feedback signals from the transport pathway.
Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II
Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purifie...
Endoplasmic reticulum stress and diabetic retinopathy
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Toshiyuki Oshitari
Full Text Available Toshiyuki Oshitari1,2, Natsuyo Hata1, Shuichi Yamamoto11Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba City, Chiba, Japan; 2Department of Ophthalmology, Kimitsu Central Hospital, Kisarazu City, Chiba, JapanAbstract: Endoplasmic reticulum (ER stress is involved in the pathogenesis of several diseases including Alzheimer disease and Parkinson disease. Many recent studies have shown that ER stress is related to the pathogenesis of diabetes mellitus, and with the death of pancreatic β-cells, insulin resistance, and the death of the vascular cells in the retina. Diabetic retinopathy is a major complication of diabetes and results in death of both neural and vascular cells. Because the death of the neurons directly affects visual function, the precise mechanism causing the death of neurons in early diabetic retinopathy must be determined. The ideal therapy for preventing the onset and the progression of diabetic retinopathy would be to treat the factors involved with both the vascular and neuronal abnormalities in diabetic retinopathy. In this review, we present evidence that ER stress is involved in the death of both retinal neurons and vascular cells in diabetic eyes, and thus reducing or blocking ER stress may be a potential therapy for preventing the onset and the progression of diabetic retinopathy.Keywords: endoplasmic reticulum stress, diabetic retinopathy, vascular cell death, neuronal cell death
Vesicular trafficking of incoming human papillomavirus 16 to the Golgi apparatus and endoplasmic reticulum requires γ-secretase activity.
Zhang, Wei; Kazakov, Teymur; Popa, Andreea; DiMaio, Daniel
The route taken by papillomaviruses from the cell surface to the nucleus during infection is incompletely understood. Here, we developed a novel human papillomavirus 16 (HPV16) pseudovirus in which the carboxy terminus of the minor capsid protein L2 is exposed on the exterior of the intact capsid prior to cell binding. With this pseudovirus, we used the proximity ligation assay immune detection technique to demonstrate that during entry HPV16 L2 traffics into and out of the early endosome prior to Golgi localization, and we demonstrated that L2 enters the endoplasmic reticulum during entry. The cellular membrane-associated protease, γ-secretase, is required for infection by HPV16 pseudovirus and authentic HPV16. We also showed that inhibition of γ-secretase does not interfere substantively with virus internalization, initiation of capsid disassembly, entry into the early endosome, or exit from this compartment, but γ-secretase is required for localization of L2 and viral DNA to the Golgi apparatus and the endoplasmic reticulum. These results show that incoming HPV16 traffics sequentially from the cell surface to the endosome and then to the Golgi apparatus and the endoplasmic reticulum prior to nuclear entry. The human papillomaviruses are small nonenveloped DNA viruses responsible for approximately 5% of all human cancer deaths, but little is known about the process by which these viruses transit from the cell surface to the nucleus. Here we show that incoming HPV16, the most common high-risk HPV, traffics though a series of vesicular compartments during infectious entry, including the endosome, Golgi apparatus, and endoplasmic reticulum. Furthermore, we show that γ-secretase, a cellular membrane-associated protease, is required for entry of the L2 minor capsid protein and viral DNA into the Golgi apparatus and endoplasmic reticulum. These studies reveal a new pathway of cell entry by DNA viruses and suggest that components of this pathway are candidate
Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics
Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio
Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245
Targeting endoplasmic reticulum stress in liver disease.
Wu, Fa-Ling; Liu, Wen-Yue; Van Poucke, Sven; Braddock, Martin; Jin, Wei-Min; Xiao, Jian; Li, Xiao-Kun; Zheng, Ming-Hua
The accumulation of unfolded protein in the endoplasmic reticulum (ER) initiates an unfolded protein response (UPR) via three signal transduction cascades, which involve protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme-1α (IRE1α) and activating transcription factor-6α (ATF6α). An ER stress response is observed in nearly all physiologies related to acute and chronic liver disease and therapeutic targeting of the mechanisms implicated in UPR signaling have attracted considerable attention. This review focuses on the correlation between ER stress and liver disease and the possible targets which may drive the potential for novel therapeutic intervention. Expert Commentary: We describe pathways which are involved in UPR signaling and their potential correlation with various liver diseases and underlying mechanisms which may present opportunities for novel therapeutic strategies are discussed.
Endoplasmic-Reticulum Calcium Depletion and Disease
Mekahli, Djalila; Bultynck, Geert; Parys, Jan B.; De Smedt, Humbert; Missiaen, Ludwig
The endoplasmic reticulum (ER) as an intracellular Ca2+ store not only sets up cytosolic Ca2+ signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca2+ depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca2+ may no longer sustain essential cell functions. On the other hand, loss of luminal Ca2+ causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca2+ depletion. PMID:21441595
Endoplasmic Reticulum Stress and Ethanol Neurotoxicity
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Fanmuyi Yang
Full Text Available Ethanol abuse affects virtually all organ systems and the central nervous system (CNS is particularly vulnerable to excessive ethanol exposure. Ethanol exposure causes profound damages to both the adult and developing brain. Prenatal ethanol exposure induces fetal alcohol spectrum disorders (FASD which is associated with mental retardation and other behavioral deficits. A number of potential mechanisms have been proposed for ethanol-induced brain damage; these include the promotion of neuroinflammation, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, and thiamine deficiency. The endoplasmic reticulum (ER regulates posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and induces unfolded protein response (UPR which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK, inositol-requiring enzyme 1 (IRE1, and activating transcription factor 6 (ATF6. UPR is initiated to protect cells from overwhelming ER protein loading. However, sustained ER stress may result in cell death. ER stress has been implied in various CNS injuries, including brain ischemia, traumatic brain injury, and aging-associated neurodegeneration, such as Alzheimer's disease (AD, Huntington's disease (HD, Amyotrophic lateral sclerosis (ALS, and Parkinson's disease (PD. However, effects of ethanol on ER stress in the CNS receive less attention. In this review, we discuss recent progress in the study of ER stress in ethanol-induced neurotoxicity. We also examine the potential mechanisms underlying ethanol-mediated ER stress and the interaction among ER stress, oxidative stress and autophagy in the context of ethanol neurotoxicity.
SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.
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Kèvin Knoops
Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.
Isolation of plasma membrane-associated membranes from rat liver.
Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R
Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.
Mitochondria-Endoplasmic Reticulum Contact Sites Mediate Innate Immune Responses.
Misawa, Takuma; Takahama, Michihiro; Saitoh, Tatsuya
Mitochondria and the endoplasmic reticulum (ER) are fundamental organelles that coordinate high-order cell functions. Mitochondria are centers of energy production, whereas the ER is responsible for folding, transport, and degradation of proteins. In addition to their specific functions, mitochondria and ER actively communicate with each other to promote a variety of cellular events, such as material transfer and signal transduction. Recent studies have shown the critical involvement of these organelles in regulation of the innate immune system, which functions in host defense. The innate immune system utilizes a wide range of germ-line-encoded pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and induces inflammatory and antiviral responses. Contact sites between mitochondria and the ER function in assembly of the NLR family pyrin domain containing 3 (NLRP3)-inflammasome to promote the inflammatory response. The NLRP3-inflammasome is a protein complex composed of the receptor NLRP3 on the ER side and the adaptor apoptosis-associated speck-like protein containing a CARD on the mitochondrial side; it induces caspase-1-dependent maturation of proinflammatory cytokines such as interleukin (IL)-1β and IL-18. Furthermore, ER-mitochondria contact sites function in initiation and mediation of signal transduction pathways downstream of intracellular PRRs, such as retinoic acid-inducible gene I-like receptor and cyclic GMP-AMP synthase, to promote the antiviral response. Therefore, ER-mitochondria contact sites, also known as mitochondria-associated membranes, play key roles in regulation of innate immune responses.
Endoplasmic reticulum stress sensing in the unfolded protein response
National Research Council Canada - National Science Library
Gardner, Brooke M; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M; Walter, Peter
Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation...
Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.
Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C
Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.
Endoplasmic Reticulum Stress in Skeletal Muscle Homeostasis and Disease
Rayavarapu, Sree; Coley, William; Nagaraju, Kanneboyina
Our appreciation of the role of endoplasmic reticulum(ER) stress pathways in both skeletal muscle homeostasis and the progression of muscle diseases is gaining momentum. This review provides insight into ER stress mechanisms during physiologic and pathological disturbances in skeletal muscle. The role of ER stress in the response to dietary alterations and acute stressors, including its role in autoimmune and genetic muscle disorders, has been described. Recent studies identifying ER stress m...
Quantification of plasmodesmatal endoplasmic reticulum coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching
DEFF Research Database (Denmark)
Martens, Helle; Roberts, Alison G.; Oparka, Karl J.
Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein...... retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome...... and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues...
Endoplasmic reticulum stress implicated in chronic traumatic encephalopathy.
Lucke-Wold, Brandon P; Turner, Ryan C; Logsdon, Aric F; Nguyen, Linda; Bailes, Julian E; Lee, John M; Robson, Matthew J; Omalu, Bennet I; Huber, Jason D; Rosen, Charles L
Chronic traumatic encephalopathy is a progressive neurodegenerative disease characterized by neurofibrillary tau tangles following repetitive neurotrauma. The underlying mechanism linking traumatic brain injury to chronic traumatic encephalopathy has not been elucidated. The authors investigate the role of endoplasmic reticulum stress as a link between acute neurotrauma and chronic neurodegeneration. The authors used pharmacological, biochemical, and behavioral tools to assess the role of endoplasmic reticulum stress in linking acute repetitive traumatic brain injury to the development of chronic neurodegeneration. Data from the authors' clinically relevant and validated rodent blast model were compared with those obtained from postmortem human chronic traumatic encephalopathy specimens from a National Football League player and World Wrestling Entertainment wrestler. The results demonstrated strong correlation of endoplasmic reticulum stress activation with subsequent tau hyperphosphorylation. Various endoplasmic reticulum stress markers were increased in human chronic traumatic encephalopathy specimens, and the endoplasmic reticulum stress response was associated with an increase in the tau kinase, glycogen synthase kinase-3β. Docosahexaenoic acid, an endoplasmic reticulum stress inhibitor, improved cognitive performance in the rat model 3 weeks after repetitive blast exposure. The data showed that docosahexaenoic acid administration substantially reduced tau hyperphosphorylation (t = 4.111, p chronic traumatic encephalopathy. Docosahexaenoic acid therefore warrants further investigation as a potential therapeutic agent for the prevention of chronic traumatic encephalopathy.
Endoplasmic Reticulum Stress and Apoptosis Mechanisms
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G.Seyda Seydel
Full Text Available Thhe endoplasmic reticulum (ER is the principal site for the synthesis, folding and maturation of most secreted proteins in the cell and essential for most cellular activities. Conditions that impair the folding capacity of the ER cause ER stress and induce a set of signaling pathways termed the unfolded protein response (UPR. If the protective mechanism activated by the UPR is not sufficient to restore normal ER function, cells die by apoptosis mechanism. Recent research suggests that ER stress and UPR play important roles in the metabolic disorders such as diabetes and obesity, cancer, immune response and in some neurodegenerative diseases such as Alzheimer, Parkinson, Huntington. Further studies on ER stress and UPR are necessary to clarify the exact role of this physiological mechanism and provide novel avenues to potential therapies. This review will provide an overview of ER stress, the UPR signaling pathways and ER stress induced apoptosis mechanism. [Archives Medical Review Journal 2012; 21(4.000: 221-235
Endoplasmic Reticulum Stress in Reproductive Function
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Kang-sheng LIU
Full Text Available Normal folding requires that unique conditions should be maintained within the endoplasmic reticulum (ER lumen, and nascent proteins are initially bound to Ca2+dependent chaperone proteins. Proteins synthesized in the ER are properly folded with the assistance of ER chaperones. misfolded proteins are disposed by ER-associated protein degradation. Accumulation of misfolded proteins in the ER triggers an adaptive ER stress response, which leads to activation of the unfolded protein response (UPR, a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cells. It has been shown that ER stress involves in pathophysiological development of many diseases, including neurological diseases. However, nowadays, a few studies have begun to focus on the possibility that the accumulation of misfolded proteins can also contribute to reproductive diseases. In this article, we mainly introduced the involvement of ER stress response in preimplantation embryos, placental development, intrauterine growth restriction (IUGR and testicular germ cells so as to provide important insights for the molecular mechanisms of ER stress-induced apoptosis in reproductive diseases.
Endoplasmic reticulum proteostasis impairment in aging.
Martínez, Gabriela; Duran-Aniotz, Claudia; Cabral-Miranda, Felipe; Vivar, Juan P; Hetz, Claudio
Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one-third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
Endoplasmic Reticulum Stress and Pancreatic Cancer
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Kemal Ergin
Full Text Available Endoplasmic reticulum (ER stress, which results from different stimuli, is an important cellular event. There are different types of response to ER stress. One of them is evolutionarily conserved unfolded protein response (UPR. UPR has three sensors for further activation of molecules. These sensors are inositol-requiring enzyme 1 (IRE1, activated transcription factor 6 (ATF6, and ER-resident protein kinase RNA (PKR-like ER kinase (PERK. In the absence of ER stress, these sensors are maintained in an inactive state. However, under ER stress conditions, they became activated and induce the downstream targets. As a consequence of ER stress, the cell may stay alive or became dead. Several studies have shown that ER stress is associated with different types of diseases such as diabetes mellitus, Alzheimer's disease, prion disease, and cancer. As a cancer type, it has been shown that pancreatic cancer is also associated with ER stress. Pancreatic cancer has a low cure potential with its late diagnosis. Its association with ER stress is seen as a new therapeutic approach. The aim of this is review is to provide an overview of the mechanisms of ER stress and its relationship with pancreatic cancer, one of the diseases in which ER stress affects pathogenesis.
Estradiol up-regulates L-type Ca2+channels via membrane-bound estrogen receptor / Phosphoinositide-3kinase / Akt / cAMP response element-binding protein signaling pathway.
Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy
In long QT type-2 (LQT2), women are more prone to lethal arrhythmias called Torsade de Pointes (TdP) than men. We previously reported that 17-β-estradiol (E2) upregulates L-type Ca 2+ -channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classical genomic-mechanism mediated by estrogen-receptor-α (ER)α. In LQT2 ( I Kr -blockade or bradycardia), the higher Ca 2+ influx via I Ca,L , causes Ca 2+ -overload, spontaneous sarcoplasmic reticulum Ca 2+ -release, and re-activation of I Ca,L that trigger early afterdepolarizations (EADs) and TdP. The molecular mechanisms whereby E2 upregulates I Ca,L are poorly understood and are now investigated. H9C2 and rat myocytes were incubated with E2, ±ER antagonist, or inhibitors of downstream transcription factors 24 hours, followed by Western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10∼100 nM) increased I Ca,L density and Cav1.2α1C expression which were suppressed by the ER-antagonist ICI-182,780 (1μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of Pi3K (LY294002=30μM; pL via plasma-membrane ER, and activating a Pi3K, Akt and CREB signaling. The promoter regions of CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding-sites for p-CREB and ERα which suggest a synergistic regulation by these pathways. Copyright © 2018. Published by Elsevier Inc.
Formation and Regulation of Mitochondrial Membranes
Directory of Open Access Journals (Sweden)
Laila Cigana Schenkel
Full Text Available Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases.
Sensory Neurons Derived from Diabetic Rats Have Diminished Internal Ca2+ Stores Linked to Impaired Re-uptake by the Endoplasmic Reticulum
Directory of Open Access Journals (Sweden)
Elena Zherebitskaya
Full Text Available Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca2+ signalling. Previous work has described abnormalities in Ca2+ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca2+ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. DRG (dorsal root ganglia sensory neurons from age-matched normal and 3–5-month-old STZ (streptozotocin-diabetic rats (an experimental model of Type 1 diabetes were cultured. At 1–2 days in vitro an array of parameters were measured to investigate Ca2+ homoeostasis including (i axonal levels of intracellular Ca2+, (ii Ca2+ uptake by the ER (endoplasmic reticulum, (iii assessment of Ca2+ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl- and caffeine-induced Ca2+ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca2+ into the ER was reduced by 2-fold (P<0.05 in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca2+ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca2+ homoeostasis possibly triggered by suboptimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.
Biogenesis of plasma membrane cholesterol
Energy Technology Data Exchange (ETDEWEB)
Lange, Y.
A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from (/sup 3/H) acetate moved to the plasma membrane with a half-time of 1 h at 37/sup 0/C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane.
The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterization of the carbonate-washed membrane standard
Peng, Lifeng; Kapp, Eugene A.; Fenyö, David; Kwon, Min-Seok; Jiang, Pu; Wu, Songfeng; Jiang, Ying; Aguilar, Marie-Isabel; Ahmed, Nikhat; Baker, Mark S.; Cai, Zongwei; Chen, Yu-Ju; Van Chi, Phan; Chung, Maxey C. M.; He, Fuchu; Len, Alice C. L.; Liao, Pao-Chi; Nakamura, Kazuyuki; Ngai, Sai Ming; Paik, Young-Ki; Pan, Tai-Long; Poon, Terence C. W.; Salekdeh, Ghasem Hosseini; Simpson, Richard J.; Sirdeshmukh, Ravi; Srisomsap, Chantragan; Svasti, Jisnuson; Tyan, Yu-Chang; Dreyer, Florian S.; McLauchlan, Danyl; Rawson, Pisana; Jordan, T. William
The Asia Oceania Human Proteome Organisation has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multi-laboratory project is based on analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. Here we present the strategy used for preparation and initial characterisation of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of seventeen independent datasets from five types of proteomic workflows is in progress. PMID:20486120
Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.
LENUS (Irish Health Repository)
Samali, Afshin
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.
Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
Echevarría, Wihelma; Leite, M Fatima; Guerra, Mateus T; Zipfel, Warren R; Nathanson, Michael H
Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.
Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome.
Campbell-Valois, François-Xavier; Trost, Matthias; Chemali, Magali; Dill, Brian D; Laplante, Annie; Duclos, Sophie; Sadeghi, Shayan; Rondeau, Christiane; Morrow, Isabel C; Bell, Christina; Gagnon, Etienne; Hatsuzawa, Kiyokata; Thibault, Pierre; Desjardins, Michel
Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.
Endoplasmic reticulum oxidoreductin 1α mediates hepatic endoplasmic reticulum stress in homocysteine-induced atherosclerosis.
Yang, Xiaoling; Xu, Hua; Hao, Yinju; Zhao, Li; Cai, Xin; Tian, Jue; Zhang, Minghao; Han, Xuebo; Ma, Shengchao; Cao, Jun; Jiang, Yideng
Endoplasmic reticulum (ER) stress is emerging as an important modulator of different pathological process and as a mechanism contributing to homocysteine (Hcy)-induced hepar injury. However, the molecular event that Hcy-induced ER stress in the hepar under the atherosclerosis background is currently unknown. Endoplasmic reticulum oxidoreductin 1α (ERO1α) plays a crucial role in maintaining ER stress function. In this study, we determined the expression of ERO1α in the hepar in hyperhomocysteinemia and the effect of ERO1α in hepacytes ER stress in the presence of Hcy. HHcy model was established by feeding the methionine diet in apolipoprotein-E-deficient (ApoE-/-) mice, and the hepatocytes were incubated with folate and different concentrations of Hcy. Our results showed that Hcy triggered ER stress characterized by an increased contents of glucose-regulated protein 78 (GRP78), protein kinase RNA-like ER kinase (PERK), activating transcription factor (ATF) 6 and X-box binding protein-1 (XBP-1). The ERO1α expressions in HHcy mice and Hcy-treated hepatocytes were decreased compared with those in ApoE-/- group and control hepacytes (P stress. Meanwhile, the expressions of ER stress-related factor including GRP78, PERK, ATF6 and XBP-1, were significantly decreased when the ERO1α gene was over-expressed in hepacytes. Our results suggested that ERO1α may be involved in Hcy-induced hepar ER stress, and the inhibition of ERO1α expression can accelerate this process. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology
Guzel, Elif; Arlier, Sefa; Guzeloglu-Kayisli, Ozlem; Tabak, Mehmet Selcuk; Ekiz, Tugba; Semerci, Nihan; Larsen, Kellie; Schatz, Frederick; Lockwood, Charles Joseph; Kayisli, Umit Ali
The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition
Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics.
Smirle, Jeffrey; Au, Catherine E; Jain, Michael; Dejgaard, Kurt; Nilsson, Tommy; Bergeron, John
Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.
Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p
Sherwood, Peter W.; Carlson, Marian
Mutations in the GSF2 gene cause glucose starvation phenotypes in Saccharomyces cerevisiae. We have isolated the HXT1 gene, which encodes a low-affinity, high-capacity glucose transporter, as a multicopy suppressor of a gsf2 mutation. We show that gsf2 mutants accumulate Hxt1p in the endoplasmic reticulum (ER) and that Gsf2p is a 46-kDa integral membrane protein localized to the ER. gsf2 mutants also display a galactose growth defect and abnormal localization of the galactose transporter Gal2...
Deficiency of Suppressor Enhancer Lin12 1 Like (SEL1L) in Mice Leads to Systemic Endoplasmic Reticulum Stress and Embryonic Lethality*
Francisco, Adam B; Singh, Rajni; Li, Shuai; Vani, Anish K.; Yang, Liu; Munroe, Robert J.; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C; Long, Qiaoming
Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted protei...
PERK and filamin A in actin cytoskeleton remodeling at ER-plasma membrane contact sites.
van Vliet, Alexander R; Agostinis, Patrizia
The endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK) plays a major role during the unfolded protein response (UPR), mainly through eIF2α phosphorylation. We uncovered that PERK, by interacting with Filamin A, elicits F-actin remodeling required for ER-plasma membrane contact site formation after ER-Ca(2+) depletion, through a UPR-independent mechanism.
DEFF Research Database (Denmark)
Bendix, Pól Martin
Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...
Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum.
Cross, Benedict C S; McKibbin, Craig; Callan, Anna C; Roboti, Peristera; Piacenti, Michela; Rabu, Catherine; Wilson, Cornelia M; Whitehead, Roger; Flitsch, Sabine L; Pool, Martin R; High, Stephen; Swanton, Eileithyia
Production and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-associated degradation (ERAD) eeyarestatin 1 (ES(I)) is a potent inhibitor of protein translocation. We have characterised this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ES(I) targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ES(I) acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ES(I), and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.
Endoplasmic reticulum stress in obesity and obesity-related disorders: An expanded view.
Pagliassotti, Michael J; Kim, Paul Y; Estrada, Andrea L; Stewart, Claire M; Gentile, Christopher L
The endoplasmic reticulum (ER) is most notable for its central roles in calcium ion storage, lipid biosynthesis, and protein sorting and processing. By virtue of its extensive membrane contact sites that connect the ER to most other organelles and to the plasma membrane, the ER can also regulate diverse cellular processes including inflammatory and insulin signaling, nutrient metabolism, and cell proliferation and death via a signaling pathway called the unfolded protein response (UPR). Chronic UPR activation has been observed in liver and/or adipose tissue of dietary and genetic murine models of obesity, and in human obesity and non-alcoholic fatty liver disease (NAFLD). Activation of the UPR in obesity and obesity-related disorders likely has two origins. One linked to classic ER stress involving the ER lumen and one linked to alterations to the ER membrane environment. This review discusses both of these origins and also considers the role of post-translational protein modifications, such as acetylation and palmitoylation, and ER-mitochondrial interactions to obesity-mediated impairments in the ER and activation of the UPR. Copyright © 2016 Elsevier Inc. All rights reserved.
Lack of cortical endoplasmic reticulum protein Ist2 alters sodium accumulation in Saccharomyces cerevisiae cells.
Papouskova, Klara; Andrsova, Marketa; Sychrova, Hana
The maintenance of intracellular alkali-metal-cation homeostasis is a fundamental property of all living organisms, including the yeast Saccharomyces cerevisiae. Several transport systems are indispensable to ensure proper alkali-metal-cation levels in the yeast cytoplasm and organelles. Ist2 is an endoplasmic reticulum (ER)-resident protein involved, together with other tethering proteins, in the formation of contacts between the plasma and ER membranes. As IST2 gene deletion was shown to influence yeast growth in the presence of sodium, we focused on the roles of Ist2 in the cell response to the presence of various concentrations of alkali metal cations, and its interactions with characterised plasma membrane alkali-metal-cation transporters. Most importantly, we show that, in BY4741 background, the lack of Ist2 results in the accumulation of higher amounts of sodium when the cells are exposed to the presence of this cation, demonstrating the importance of Ist2 for the maintenance of low intracellular levels of toxic sodium. As the function and localisation of alkali-metal-cation exporters is not affected in ist2Δ cells, IST2 deletion results in an increased non-specific uptake of sodium to cells. Moreover, the deletion of IST2 influences relative cell membrane potential, pHin and the growth of cells in the presence of a limiting K+ concentration. © FEMS 2017. All rights reserved. For permissions, please e-mail: email@example.com.
Layer-by-layer cell membrane assembly
Matosevic, Sandro; Paegel, Brian M.
Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.
Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.
Directory of Open Access Journals (Sweden)
Cédric Lenormand
Full Text Available Langerin is required for the biogenesis of Birbeck granules (BGs, the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER, with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These
The control of the contraction of myocytes from guinea-pig heart by the resting membrane potential.
Mermi, J.; Yajima, M.; Ebner, F.
1. The influence of different holding potentials (-120 to -70 mV) on the contraction of enzymatically dispersed myocytes from guinea-pig hearts was evaluated. Contractions were elicited by repetitive depolarizations to 0 mV at 0.5 Hz. 2. While ineffective at 140 and 5 mmol l-1 [Na+]o and pipette Na+, respectively, depolarization of the resting membrane with the holding potential increased myocyte shortening at reduced Na+ gradients ([Na+]o 70 or [Na+]i 10-15 mmol l-1). Elevated intracellular Na+ after Na(+)-pump inhibition with ouabain 1-10 mumol l-1 was similarly effective with regard to the inotropic response to different holding potentials. 3. At -70 mV holding potential, reduction of [Na+]o from 140 to 70 mmol l-1 increased myocyte shortening and induced an inwardly directed component of the holding current which peaked at -44 +/- 10 pA and declined thereafter in parallel with the inotropic effect. The relation of this inward current to [Ca2+]i was confirmed by experiments at high Ca2+ buffer capacity where [Na+]o reduction induced a Ni(2+)-insensitive, outwardly directed component (36 +/- 15 pA) of the holding current. The observed inward current is suggested to reflect the extrusion of [Ca2+]i in exchange for [Na+]o as a counter-regulatory mechanism which limits the increase of [Ca2+]i. 4. The interventions which increased the strength of the contraction also enhanced the transient tail current after repolarization, suggesting its close relation to [Ca2+]i. This finding confirmed the pattern found with cell shortening. 5. It is concluded that under certain conditions, voltage-dependent and Na(+)-dependent Na(+)-Ca2+ exchange during the interval between the contractions is relevant to the diastolic concentration of [Ca2+]i which in turn determines the accumulation of Ca2+ in the sarcoplasmic reticulum and the magnitude of the subsequent contraction. PMID:1797330
Region-specific vulnerability to endoplasmic reticulum stress ...
Indian Academy of Sciences (India)
Nov 6, 2013 ... Region-specific vulnerability to endoplasmic reticulum stress-induced neuronal death in rat brain after status epilepticus. JING CHEN. †,*. , HU GUO. †. , GUO ZHENG and ZHONG-NAN SHI. Department of Neurology, Nanjing Children's Hospital Affiliated to Nanjing Medical. University, No. 72, Guangzhou ...
How are proteins reduced in the endoplasmic reticulum?
DEFF Research Database (Denmark)
Ellgaard, Lars; Sevier, Carolyn S.; Bulleid, Neil J.
The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction o...
Effect of tumour necrosis factor-alpha on total myofibrillar and sarcoplasmic protein synthesis and polysomal aggregation in rat skeletal muscles.
Cheema, I R; Hermann, C; Postell, S; Holifield, B
The total sarcoplasmic and myofibrillar protein synthesis was reduced in incubated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus of rat after in vivo tumour necrosis factor-alpha treatment at 50 micrograms/kg/day for 5 days. The rate of protein synthesis in the myofibrillar fraction was inhibited more severely (41% in EDL and 34% in soleus) than that in the sarcoplasmic fraction (23% in EDL and 14% in soleus). Sucrose density gradient centrifugation analysis indicated that TNF-alpha treatment impaired polysomal aggregation in rat diaphragm muscle. Compared with the control muscles, the ratio of 40S and 60S subunits to polysomes was higher in TNF-alpha treated muscles. These findings suggest a role for TNF-alpha in the translational regulation of protein synthesis in rat skeletal muscle.
Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences
DEFF Research Database (Denmark)
Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H
phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...
UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum*
Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D.; Tremblay, Michel L.; Michalak, Marek
Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. PMID:25586181
UBC9-dependent association between calnexin and protein tyrosine phosphatase 1B (PTP1B) at the endoplasmic reticulum.
Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D; Tremblay, Michel L; Michalak, Marek
Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum
Caesar, Katharina; Thamm, Antje M. K.; Witthöft, Janika; Elgass, Kirstin; Huppenberger, Peter; Grefen, Christopher; Horak, Jakub; Harter, Klaus
Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants. PMID:21841169
Citrate synthase, sarcoplasmic reticular calcium ATPase, and choline acetyltransferase activities of specific pelvic floor muscles of the rabbit.
Spettel, Sara; De, Elise; Elias, Tamer; Schuler, Catherine; Leggett, Robert E; Levin, Robert M
There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic-endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity.
The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm
DEFF Research Database (Denmark)
Winther, Anne-Marie Lund; Bublitz, Maike; Karlsen, Jesper Lykkegaard
The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal....... Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called...... report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure...
Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming
Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki
ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly
NETWORKED 3B: a novel protein in the actin cytoskeleton-endoplasmic reticulum interaction.
Wang, Pengwei; Hussey, Patrick J
In plants movement of the endoplasmic reticulum (ER) is dependent on the actin cytoskeleton. However little is known about proteins that link the ER membrane and the actin cytoskeleton. Here we identified a novel protein, NETWORKED 3B (NET3B), which is associated with the ER and actin cytoskeleton in vivo. NET3B belongs to a superfamily of plant specific actin binding proteins, the NETWORKED family. NET3B associates with the actin cytoskeleton in vivo through an N-terminal NET actin binding (NAB) domain, which has been well-characterized in other members of the NET family. A three amino acid insertion, Val-Glu-Asp, in the NAB domain of NET3B appears to lower its ability to localize to the actin cytoskeleton compared with NET1A, the founding member of the NET family. The C-terminal domain of NET3B links the protein to the ER. Overexpression of NET3B enhanced the association between the ER and the actin cytoskeleton, and the extent of this association was dependent on the amount of NET3B available. Another effect of NET3B overexpression was a reduction in ER membrane diffusion. In conclusion, our results revealed that NET3B modulates ER and actin cytoskeleton interactions in higher plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
The Ca(2+)-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.
Espinoza-Fonseca, L Michel
Ca(2+) transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca(2+) homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca(2+) uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca(2+) and other ions across the SR. During Ca(2+) uptake by the SR Ca(2+)-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca(2+) transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca(2+) release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca(2+) transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.
Membrane charge moved at contraction thresholds in skeletal muscle fibres.
Horowicz, P; Schneider, M F
(Q) time courses could be reproduced. The best theoretical records obtained with n = 3 still passed below the shoulders, second rising phases and later parts of the early constant phases in the various experimental I(Q) records. Theoretical records did fit accurately the I(Q) time courses observed under steady partial fibre depolarization. The relatively small current not reproduced by the model may be an electrical accompaniment of the activation of calcium release or the elevation of internal free calcium levels in the space between the transverse tubules (T-tubules) and the sarcoplasmic reticulum.
PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy.
Salaroglio, Iris C; Panada, Elisa; Moiso, Enrico; Buondonno, Ilaria; Provero, Paolo; Rubinstein, Menachem; Kopecka, Joanna; Riganti, Chiara
Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype. To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells. ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts. Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.
The endoplasmic reticulum stress response: A link with tuberculosis?
Cui, Yongyong; Zhao, Deming; Barrow, Paul Andrew; Zhou, Xiangmei
Tuberculosis (TB) remains a major cause of mortality and morbidity in the worldwide. The endoplasmic-reticulum stress (ERS) response constitutes a cellular process that is triggered by mycobacterial infection that disturbs the folding of proteins in the endoplasmic reticulum (ER). The unfolded protein response (UPR) is induced to suspend the synthesis of early proteins and reduce the accumulation of unfolded- or misfolded proteins in the ER restoring normal physiological cell function. Prolonged or uncontrolled ERS leads to the activation of three signaling pathways (IRE1, PERK and ATF6) which directs the cell towards apoptosis. The absence of this process facilitates spread of the mycobacteria within the body. We summarize here recent advances in understanding the signaling pathway diversity governing ERS in relation to TB. Copyright © 2015 Elsevier Ltd. All rights reserved.
Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?
DEFF Research Database (Denmark)
Skagen, Peter Storgaard; Horn, T; Kruse, H A
Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities...... in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in "bundles" and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae...... polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification....
Membrane tension and membrane fusion
Kozlov, Michael M.; Chernomordik, Leonid V.
Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually la...
Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?
DEFF Research Database (Denmark)
Skagen, Peter Storgaard; Horn, T; Kruse, H A
and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non-conclusive, only single nucleotide...... polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification....
Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.
Zhang, Ying; Zhang, KaiQiang; Ma, Lin; Gu, HeFeng; Li, Jian; Lei, Shuang
The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.
Prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine
Directory of Open Access Journals (Sweden)
Ravindra Kumar
Full Text Available Background The endoplasmic reticulum plays an important role in many cellular processes, which includes protein synthesis, folding and post-translational processing of newly synthesized proteins. It is also the site for quality control of misfolded proteins and entry point of extracellular proteins to the secretory pathway. Hence at any given point of time, endoplasmic reticulum contains two different cohorts of proteins, (i proteins involved in endoplasmic reticulum-specific function, which reside in the lumen of the endoplasmic reticulum, called as endoplasmic reticulum resident proteins and (ii proteins which are in process of moving to the extracellular space. Thus, endoplasmic reticulum resident proteins must somehow be distinguished from newly synthesized secretory proteins, which pass through the endoplasmic reticulum on their way out of the cell. Approximately only 50% of the proteins used in this study as training data had endoplasmic reticulum retention signal, which shows that these signals are not essentially present in all endoplasmic reticulum resident proteins. This also strongly indicates the role of additional factors in retention of endoplasmic reticulum-specific proteins inside the endoplasmic reticulum. Methods This is a support vector machine based method, where we had used different forms of protein features as inputs for support vector machine to develop the prediction models. During training leave-one-out approach of cross-validation was used. Maximum performance was obtained with a combination of amino acid compositions of different part of proteins. Results In this study, we have reported a novel support vector machine based method for predicting endoplasmic reticulum resident proteins, named as ERPred. During training we achieved a maximum accuracy of 81.42% with leave-one-out approach of cross-validation. When evaluated on independent dataset, ERPred did prediction with sensitivity of 72.31% and specificity of 83
Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis
Kaser, Arthur; Martínez-Naves, Eduardo; Blumberg, Richard S.
Purpose of review To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). Recent findings The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. Summary Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders. PMID:20495455
Formation and Stability of Lipid Membrane Nanotubes.
Bahrami, Amir Houshang; Hummer, Gerhard
Lipid membrane nanotubes are abundant in living cells, even though tubules are energetically less stable than sheet-like structures. According to membrane elastic theory, the tubular endoplasmic reticulum (ER), with its high area-to-volume ratio, appears to be particularly unstable. We explore how tubular membrane structures can nevertheless be induced and why they persist. In Monte Carlo simulations of a fluid-elastic membrane model subject to thermal fluctuations and without constraints on symmetry, we find that a steady increase in the area-to-volume ratio readily induces tubular structures. In simulations mimicking the ER wrapped around the cell nucleus, tubules emerge naturally as the membrane area increases. Once formed, a high energy barrier separates tubules from the thermodynamically favored sheet-like membrane structures. Remarkably, this barrier persists even at large area-to-volume ratios, protecting tubules against shape transformations despite enormous driving forces toward sheet-like structures. Molecular dynamics simulations of a molecular membrane model confirm the metastability of tubular structures. Volume reduction by osmotic regulation and membrane area growth by lipid production and by fusion of small vesicles emerge as powerful factors in the induction and stabilization of tubular membrane structures.
Momordicacharantia: A New Strategic Vision to Improve the Therapy of Endoplasmic Reticulum Stress.
Chong, Wai Chin; Gundamaraju, Rohit; Vemuri, Ravichandra; Scotti, Marcus T; Scotti, Luciana
Endoplasmic reticulum is the major site for protein biosynthesis. Any perturbation in the endoplasmic reticulum will compromise its functions and reduce its efficiency in protein biosynthesis. As a result, misfolded proteins are produced and accumulated in the endoplasmic reticulum. This will result in endoplasmic reticulum stress, which reduces the quantity and quality of the functional protein synthesized from the cell. Besides that, the protein biosynthesis is also highly affected by oxidative stress. Oxidative stress is a condition where the cell has excessive exogenous and endogenous radical oxygen species overwhelming the human body's antioxidant mechanisms. Recent researches have shown that endoplasmic reticulum stress has a strong relationship with oxidative stress and both of them can form a vicious cycle that exacerbates endoplasmic reticulum stress and oxidative stress. Endoplasmic reticulum stress and oxidative stress are harmful to human health since they can disrupt cellular homoeostasis and damage cells. This work reported several studies that demonstrate that endoplasmic reticulum stress and oxidative stress have a vital role in the pathogenesis of several diseases, such as diabetes and colitis. Further research is needed to develop the therapeutic strategies that resolve endoplasmic reticulum stress and oxidative stress in order to treat these diseases. Currently, Momordicacharantia attracts worldwide attentions due to its various beneficial effects as the functional foods and can possibly help in treating endoplasmic reticulum stress and oxidative stress. Copyright© Bentham Science Publishers; For any queries, please email at email@example.com.
DEFF Research Database (Denmark)
Bendix, Pól Martin
At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....
Report on the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology: "Membrane Proteins in Health and Disease".
Reithmeier, Reinhart A F; Casey, Joseph R
The meeting "Membrane Proteins in Health and Disease" featured 6 sessions and 2 satellite meetings. At the opening session, Gunnar von Heijne delivered a plenary lecture entitled Insertion of Membrane Proteins into the Endoplasmic Reticulum. The following session topics were Membrane Protein Trafficking and Folding, Regulation of Membrane Proteins, Membrane Protein Structure, Membrane Proteins in Diverse Species, and Membrane Proteins and Diseases. The satellite meetings discussed bicarbonate transporters and Na+/H+ exchangers. Together the 21 lectures and 106 posters presented at the meeting spanned the full spectrum of current research into membrane protein structure and function.
Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest.
Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge
Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca(2+), loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest.
Urban planning of the endoplasmic reticulum (ER): how diverse mechanisms segregate the many functions of the ER.
Lynes, Emily M; Simmen, Thomas
The endoplasmic reticulum (ER) is the biggest organelle in most cell types, but its characterization as an organelle with a continuous membrane belies the fact that the ER is actually an assembly of several, distinct membrane domains that execute diverse functions. Almost 20 years ago, an essay by Sitia and Meldolesi first listed what was known at the time about domain formation within the ER. In the time that has passed since, additional ER domains have been discovered and characterized. These include the mitochondria-associated membrane (MAM), the ER quality control compartment (ERQC), where ER-associated degradation (ERAD) occurs, and the plasma membrane-associated membrane (PAM). Insight has been gained into the separation of nuclear envelope proteins from the remainder of the ER. Research has also shown that the biogenesis of peroxisomes and lipid droplets occurs on specialized membranes of the ER. Several studies have shown the existence of specific marker proteins found on all these domains and how they are targeted there. Moreover, a first set of cytosolic ER-associated sorting proteins, including phosphofurin acidic cluster sorting protein 2 (PACS-2) and Rab32 have been identified. Intra-ER targeting mechanisms appear to be superimposed onto ER retention mechanisms and rely on transmembrane and cytosolic sequences. The crucial roles of ER domain formation for cell physiology are highlighted with the specific targeting of the tumor metastasis regulator gp78 to ERAD-mediating membranes or of the promyelocytic leukemia protein to the MAM. Copyright © 2011 Elsevier B.V. All rights reserved.
[Enzymatic properties in muscle membranes].
Kursky, M D; Grigoryeva, V A
A study in the enzymatic properties of muscle membranes established that sarcolemma of the rabbit skeletal muscles contains the Ca2+-ATPase system which does not require Mg2+ for manifestation of ions activity. By some kinetic properties it differs from ATPase of myosin. The complex Ca-ATP2+ is a substrate of Ca2+-ATPase. Ions of a series of bivalent metals inhibit the latter as well as the passive transport of Ca2+, that may evidence for a definite relation of Ca2+-ATPase with Ca+2 transport in skeletal muscles. Acetyl cholinesterase and AMP-aminohydrolase are strongly bound with the sarcolemma. The sarcolemma structural organization is shown to play a certain role in manifestation of their activity. On the basis of the data obtained when studying the activity in the ATPase systems and dynamics of formation and decay of the intermediate phosphorylated product in the microsomal fraction of cow and rabbit myometrium certain peculiarities are established for the active mechanisms of Ca2+ transport in smooth muscles. A problem is under discussion on the possible active participation of sarcolemma in regulation of Ca2+ concentration in the smooth muscle cells. Two ATPase systems, Mg2+-dependent and Mg2+-dependent Ca2+ activated are found in nuclei; the role of lipids of the skeletal muscles in manifestation of their activity is studied. AMP-amino hydrolase properties are characterized for different areas of the sarcoplasmatic reticulum membranes. The model of E-avitaminous muscular distrophy was used to show disturbances in the structure of sarcolemma and membranes of the sarcoplasmatic reticulum which are accompanied by changes in their ATPase and Ca2+-transporting properties.
The ER membrane protein complex is a transmembrane domain insertase
Guna, Alina; Volkmar, Norbert; Christianson, John C.; Hegde, Ramanujan S.
Insertion of proteins into membranes is an essential cellular process. The extensive biophysical and topological diversity of membrane proteins necessitates multiple insertion pathways that remain incompletely defined. Here, we found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms. PMID:29242231
Analysis of Membrane Protein Topology in the Plant Secretory Pathway.
Guo, Jinya; Miao, Yansong; Cai, Yi
Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.
Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.
Hagemeijer, Marne C; Monastyrska, Iryna; Griffith, Janice; van der Sluijs, Peter; Voortman, Jarno; van Bergen en Henegouwen, Paul M; Vonk, Annelotte M; Rottier, Peter J M; Reggiori, Fulvio; de Haan, Cornelis A M
Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells. Copyright © 2014 Elsevier Inc. All rights reserved.
Detection and quantification of endoplasmic reticulum stress in living cells using the fluorescent compound, Thioflavin T.
Beriault, Daniel R; Werstuck, Geoff H
The endoplasmic reticulum (ER) plays a central role in the co- and post-translational modification of many proteins. Disruption of these processes can lead to the accumulation of misfolded proteins in the endoplasmic reticulum - a condition known as endoplasmic reticulum stress. In recent years, the association of endoplasmic reticulum stress with a number of disease pathologies has increased interest in the study of this condition. Current methods to detect endoplasmic reticulum stress are indirect and retrospective. Here we describe a new method to detect and quantify endoplasmic reticulum stress in live cells using Thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence when it binds to protein aggregates. We show that enhanced ThT-fluorescence correlates directly with established indicators of unfolded protein response activation. Furthermore, enhanced ThT-fluorescence can be detected in living cells within 20 min of application of an endoplasmic reticulum stress-inducing agent. ThT is capable of detecting endoplasmic reticulum stress induced by distinctly different conditions and compounds, in different cultured cell types as well as in mouse tissue samples. Pre-treatment with a potent endoplasmic reticulum stress-reducing agent, 4-phenylbutyric acid, mitigates the enhanced ThT signal. This new tool will be useful in future research investigating the role of protein misfolding in the development and/or progression of human diseases. Copyright © 2013 Elsevier B.V. All rights reserved.
Cubic membranes: a legend beyond the Flatland* of cell membrane organization.
Almsherqi, Zakaria A; Kohlwein, Sepp D; Deng, Yuru
Cubic membranes represent highly curved, three-dimensional nanoperiodic structures that correspond to mathematically well defined triply periodic minimal surfaces. Although they have been observed in numerous cell types and under different conditions, particularly in stressed, diseased, or virally infected cells, knowledge about the formation and function of nonlamellar, cubic structures in biological systems is scarce, and research so far is restricted to the descriptive level. We show that the "organized smooth endoplasmic reticulum" (OSER; Snapp, E.L., R.S. Hegde, M. Francolini, F. Lombardo, S. Colombo, E. Pedrazzini, N. Borgese, and J. Lippincott-Schwartz. 2003. J. Cell Biol. 163:257-269), which is formed in response to elevated levels of specific membrane-resident proteins, is actually the two-dimensional representation of two subtypes of cubic membrane morphology. Controlled OSER induction may thus provide, for the first time, a valuable tool to study cubic membrane formation and function at the molecular level.
[Effect of endoplasmic reticulum stress-responsive protein glucose-regulated protein 78, 94 and endoplasmic reticulum apoptosis factor caspase-12 in trophocyte on the pathogenesis of preeclampsia].
Sun, Li-Zhou; Ma, Xiao-Tian; Ge, Zhi-Ping; Han, Ping
To evaluate the relationship between pathogenesis of preeclampsia (PE) and the ultrastructure change of the endoplasmic reticulum in trophocyte, mRNA and protein expression levels of endoplasmic reticulum molecular chaperone glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), endoplasmic reticulum apoptosis factor cysteine protease protein 12 (caspase-12). Sixty-five pregnant women who were hospitalized in the First Affiliated Hospital of Nanjing Medical University from July 2008 to January 2010, were selected as the subject. Thirty pregnancy women diagnosed with PE were divided into PE group and 35 normal pregnant women were used as control group. Electron Microscopy was used to measure ultrastructure change of the endoplasmic reticulum in placenta trophocyte. Reverse transcription (RT) PCR and western blot were used to investigute the expression levels of GRP78, GRP94, caspase-12 mRNA and protein in placenta. (1) In control group the volume of endoplasmic reticulum does not increase; no swelling and no expansion of endoplasmic reticulum was found. In PE group the edema number of endoplasmic reticulum was reduced; the volume of endoplasmic reticulum increased; expansion and vacuolation of cavity and degranulation of the endoplasmic reticulum was observed significantly. (2) The mRNA and protein expression levels of GRP78 in placenta of PE group (2.59 ± 0.09 and 0.81 ± 0.31) were significantly higher than those in placenta of control group (1.16 ± 0.07 and 0.40 ± 0.10, P apoptosis may be involved in the pathophysiological processes of PE.
Acrolein induces endoplasmic reticulum stress and causes airspace enlargement.
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Yoshiaki Kitaguchi
Full Text Available BACKGROUND: Given the relative abundance and toxic potential of acrolein in inhaled cigarette smoke, it is surprising how little is known about the pulmonary and systemic effects of acrolein. Here we test the hypothesis whether systemic administration of acrolein could cause endoplasmic reticulum (ER stress, and lung cell apoptosis, leading to the enlargement of the alveolar air spaces in rats. METHODS: Acute and chronic effects of intraperitoneally administered acrolein were tested. Mean alveolar airspace area was measured by using light microscopy and imaging system software. TUNEL staining and immunohistochemistry (IHC for active caspase 3 and Western blot analysis for active caspase 3, and caspase 12 were performed to detect apoptosis. The ER-stress related gene expression in the lungs was determined by Quantitative real-time PCR analysis. Acrolein-protein adducts in the lung tissue were detected by IHC. RESULTS: Acute administration of acrolein caused a significant elevation of activated caspase 3, upregulation of VEGF expression and induced ER stress proteins in the lung tissue. The chronic administration of acrolein in rats led to emphysematous lung tissue remodeling. TUNEL staining and IHC for cleaved caspase 3 showed a large number of apoptotic septal cells in the acrolein-treated rat lungs. Chronic acrolein administration cause the endoplasmic reticulum stress response manifested by significant upregulation of ATF4, CHOP and GADd34 expression. In smokers with COPD there was a considerable accumulation of acrolein-protein adducts in the inflammatory, airway and vascular cells. CONCLUSIONS: Systemic administration of acrolein causes endoplasmic reticulum stress response, lung cell apoptosis, and chronic administration leads to the enlargement of the alveolar air spaces and emphysema in rats. The substantial accumulation of acrolein-protein adducts in the lungs of COPD patients suggest a role of acrolein in the pathogenesis of emphysema.
Emergence of the mitochondrial reticulum from fission and fusion dynamics.
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Valerii M Sukhorukov
Full Text Available Mitochondria form a dynamic tubular reticulum within eukaryotic cells. Currently, quantitative understanding of its morphological characteristics is largely absent, despite major progress in deciphering the molecular fission and fusion machineries shaping its structure. Here we address the principles of formation and the large-scale organization of the cell-wide network of mitochondria. On the basis of experimentally determined structural features we establish the tip-to-tip and tip-to-side fission and fusion events as dominant reactions in the motility of this organelle. Subsequently, we introduce a graph-based model of the chondriome able to encompass its inherent variability in a single framework. Using both mean-field deterministic and explicit stochastic mathematical methods we establish a relationship between the chondriome structural network characteristics and underlying kinetic rate parameters. The computational analysis indicates that mitochondrial networks exhibit a percolation threshold. Intrinsic morphological instability of the mitochondrial reticulum resulting from its vicinity to the percolation transition is proposed as a novel mechanism that can be utilized by cells for optimizing their functional competence via dynamic remodeling of the chondriome. The detailed size distribution of the network components predicted by the dynamic graph representation introduces a relationship between chondriome characteristics and cell function. It forms a basis for understanding the architecture of mitochondria as a cell-wide but inhomogeneous organelle. Analysis of the reticulum adaptive configuration offers a direct clarification for its impact on numerous physiological processes strongly dependent on mitochondrial dynamics and organization, such as efficiency of cellular metabolism, tissue differentiation and aging.
The role of the endoplasmic reticulum stress response following cerebral ischemia.
Hadley, Gina; Neuhaus, Ain A; Couch, Yvonne; Beard, Daniel J; Adriaanse, Bryan A; Vekrellis, Kostas; DeLuca, Gabriele C; Papadakis, Michalis; Sutherland, Brad A; Buchan, Alastair M
Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.
Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.
Reid, David W; Nicchitta, Christopher V
In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a more substantial role for the ER cellular protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression.
Endoplasmic Reticulum Stress in the Diabetic Kidney, the Good, the Bad and the Ugly
Cunard, Robyn
Diabetic kidney disease is the leading worldwide cause of end stage kidney disease and a growing public health challenge. The diabetic kidney is exposed to many environmental stressors and each cell type has developed intricate signaling systems designed to restore optimal cellular function. The unfolded protein response (UPR) is a homeostatic pathway that regulates endoplasmic reticulum (ER) membrane structure and secretory function. Studies suggest that the UPR is activated in the diabetic kidney to restore normal ER function and viability. However, when the cell is continuously stressed in an environment that lies outside of its normal physiological range, then the UPR is known as the ER stress response. The UPR reduces protein synthesis, augments the ER folding capacity and downregulates mRNA expression of genes by multiple pathways. Aberrant activation of ER stress can also induce inflammation and cellular apoptosis, and modify signaling of protective processes such as autophagy and mTORC activation. The following review will discuss our current understanding of ER stress in the diabetic kidney and explore novel means of modulating ER stress and its interacting signaling cascades with the overall goal of identifying therapeutic strategies that will improve outcomes in diabetic nephropathy. PMID:26239352
Estrogen reduces endoplasmic reticulum stress to protect against glucotoxicity induced-pancreatic β-cell death.
Kooptiwut, Suwattanee; Mahawong, Pitchnischa; Hanchang, Wanthanee; Semprasert, Namoiy; Kaewin, Suchada; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-Thai
Estrogen can improve glucose homeostasis not only in diabetic rodents but also in humans. However, the molecular mechanism by which estrogen prevents pancreatic β-cell death remains unclear. To investigate this issue, INS-1 cells, a rat insulinoma cell line, were cultured in medium with either 11.1mM or 40mM glucose in the presence or the absence of estrogen. Estrogen significantly reduced apoptotic β-cell death by decreasing nitrogen-induced oxidative stress and the expression of the ER stress markers GRP 78, ATF6, P-PERK, PERK, uXBP1, sXBP1, and CHOP in INS-1 cells after prolonged culture in medium with 40mM glucose. In contrast, estrogen increased the expression of survival proteins, including sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA-2), Bcl-2, and P-p38, in INS-1 cells after prolonged culture in medium with 40mM glucose. The cytoprotective effect of estrogen was attenuated by addition of the estrogen receptor (ERα and ERβ) antagonist ICI 182,780 and the estrogen membrane receptor inhibitor G15. We showed that estrogen decreases not only oxidative stress but also ER stress to protect against 40mM glucose-induced pancreatic β-cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.
Reactive oxygen species, endoplasmic reticulum stress and mitochondrial dysfunction: the link with cardiac arrhythmogenesis
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Gary Tse
Full Text Available Background: Cardiac arrhythmias represent a significant problem globally, leading to cerebrovascular accidents, myocardial infarction, and sudden cardiac death. There is increasing evidence to suggest that increased oxidative stress from reactive oxygen species (ROS, which is elevated in conditions such as diabetes and hypertension, can lead to arrhythmogenesis. Method: A literature review was undertaken to screen for articles that investigated the effects of ROS on cardiac ion channel function, remodelling and arrhythmogenesis. Results: Prolonged endoplasmic reticulum stress is observed in heart failure, leading to increased production of ROS. Mitochondrial ROS, which is elevated in diabetes and hypertension, can stimulate its own production in a positive feedback loop, termed ROS-induced ROS release. Together with activation, mitochondrial inner membrane anion channels, it leads to mitochondrial depolarization. Abnormal function of these organelles can then activate downstream signalling pathways, ultimately culminating in altered function or expression of cardiac ion channels responsible for generating the cardiac action potential (AP. Vascular and cardiac endothelial cells become dysfunctional, leading to altered paracrine signalling to influence the electrophysiology of adjacent cardiomyocytes. All of these changes can in turn produce abnormalities in AP repolarization or conduction, thereby increasing likelihood of triggered activity and reentry. Conclusion: ROS plays a significant role in producing arrhythmic substrate. Therapeutic strategies targeting upstream events include production of a strong reducing environment or the use of pharmacological agents that target organelle-specific proteins and ion channels. These may relieve oxidative stress and in turn prevent arrhythmic complications in patients with diabetes, hypertension and heart failure.
Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.
Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D
Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.
Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella.
Zhang, Xin; Tee, Yee Han; Heng, Justin K; Zhu, Yajuan; Hu, Xian; Margadant, Felix; Ballestrem, Christoph; Bershadsky, Alexander; Griffiths, Gareth; Yu, Hanry
Focal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.
Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.
Ahmad, Shama; Ahmad, Aftab; Hendry-Hofer, Tara B; Loader, Joan E; Claycomb, William C; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L; Chandler, Joshua D; Day, Brian J; Veress, Livia A; White, Carl W
Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.
Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity
Ahmad, Aftab; Hendry-Hofer, Tara B.; Loader, Joan E.; Claycomb, William C.; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L.; Chandler, Joshua D.; Day, Brian J.; Veress, Livia A.; White, Carl W.
Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881
Endoplasmic reticulum-mitochondrial crosstalk: a novel role for the mitochondrial peptide humanin
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Parameswaran G Sreekumar
Full Text Available In this review, the interactive mechanisms of mitochondria with the endoplasmic reticulum (ER are discussed with emphasis on the potential protective role of the mitochondria derived peptide humanin (HN in ER stress. The ER and mitochondria are dynamic organelles capable of modifying their structure and function in response to changing environmental conditions. The ER and mitochondria join together at multiple sites and form mitochondria-ER associated membranes that participate in signal transduction pathways that are under active investigation. Our laboratory previously showed that HN protects cells from oxidative stress induced cell death and more recently, described the beneficial role of HN on ER stress-induced apoptosis in retinal pigment epithelium cells and the involvement of ER-mitochondrial cross-talk in cellular protection. The protection was achieved, in part, by the restoration of mitochondrial glutathione that was depleted by ER stress. Thus, HN may be a promising candidate for therapy for diseases that involve both oxidative and ER stress. Developing novel approaches for retinal delivery of HN, its analogues as well as small molecular weight ER stress inhibitors would prove to be a valuable approach in the treatment of age-related macular degeneration.
Endoplasmic Reticulum Stress in the Diabetic Kidney, the Good, the Bad and the Ugly
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Robyn Cunard
Full Text Available Diabetic kidney disease is the leading worldwide cause of end stage kidney disease and a growing public health challenge. The diabetic kidney is exposed to many environmental stressors and each cell type has developed intricate signaling systems designed to restore optimal cellular function. The unfolded protein response (UPR is a homeostatic pathway that regulates endoplasmic reticulum (ER membrane structure and secretory function. Studies suggest that the UPR is activated in the diabetic kidney to restore normal ER function and viability. However, when the cell is continuously stressed in an environment that lies outside of its normal physiological range, then the UPR is known as the ER stress response. The UPR reduces protein synthesis, augments the ER folding capacity and downregulates mRNA expression of genes by multiple pathways. Aberrant activation of ER stress can also induce inflammation and cellular apoptosis, and modify signaling of protective processes such as autophagy and mTORC activation. The following review will discuss our current understanding of ER stress in the diabetic kidney and explore novel means of modulating ER stress and its interacting signaling cascades with the overall goal of identifying therapeutic strategies that will improve outcomes in diabetic nephropathy.
Overexpressed PLTP in macrophage may promote cholesterol accumulation by prolonged endoplasmic reticulum stress.
Yang, Xinquan; Yu, Yang; Wang, Daxin; Qin, Shucun
It is well known that phospholipid transfer protein (PLTP) is involved in the lipid metabolism and development of atherosclerosis (AS). Abundant PLTP is considered to be expressed on the foam cells derived from monocyte/macrophages in atherosclerotic plaques, suggesting that high level of active PLTP may promote the formation of foam cells. However, the exact role of PLTP on the process of macrophage derived foam cell formation remains unclear. The accumulation of free cholesterol (FC) in the cytoplasm may lead to the prolonged endoplasmic reticulum stress (ERs) and the imbalance of intracellular cholesterol homeostasis. Different PLTP level definitely alternates the phospholipids (PL) and cholesterol level in plasma, strongly suggesting that active PLTP may change the level of FC and PL intracellularly, which subsequently induced the ERs in macrophage. Thus, we hypothesize that high level of PLTP may promote the accumulation of cholesterol in macrophage via the alteration ratio of FC to PL. Therefore, validating this hypothesis may clarify the role of PLTP in macrophage ERs in AS and also raise a novel strategy in the regression of AS plaques via restoring intracellular membrane lipid homeostasis and attenuating ERs. Copyright © 2016 Elsevier Ltd. All rights reserved.
The Role of Endoplasmic Reticulum Stress in Cardiovascular Disease and Exercise
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Junyoung Hong
Full Text Available Endoplasmic reticulum (ER stress, which is highly associated with cardiovascular disease, is triggered by a disturbance in ER function because of protein misfolding or an increase in protein secretion. Prolonged disruption of ER causes ER stress and activation of the unfolded protein response (UPR and leads to various diseases. Eukaryotic cells respond to ER stress via three major sensors that are bound to the ER membrane: activating transcription factor 6 (ATF6, inositol-requiring protein 1α (IRE1α, and protein kinase RNA-like ER kinase (PERK. Chronic activation of ER stress causes damage in endothelial cells (EC via apoptosis, inflammation, and oxidative stress signaling pathways. The alleviation of ER stress has recently been accepted as a potential therapeutic target to treat cardiovascular diseases such as heart failure, hypertension, and atherosclerosis. Exercise training is an effective nonpharmacological approach for preventing and alleviating cardiovascular disease. We here review the recent viewing of ER stress-mediated apoptosis and inflammation signaling pathways in cardiovascular disease and the role of exercise in ER stress-associated diseases.
Contribution of mitochondria and endoplasmic reticulum dysfunction in insulin resistance: Distinct or interrelated roles?
Rieusset, J
Mitochondria and the endoplasmic reticulum (ER) regulate numerous cellular processes, and are critical contributors to cellular and whole-body homoeostasis. More important, mitochondrial dysfunction and ER stress are both closely associated with hepatic and skeletal muscle insulin resistance, thereby playing crucial roles in altered glucose homoeostasis in type 2 diabetes mellitus (T2DM). The accumulated evidence also suggests a potential interrelationship between alterations in both types of organelles, as mitochondrial dysfunction could participate in activation of the unfolded protein response, whereas ER stress could influence mitochondrial function. The fact that mitochondria and the ER are physically and functionally interconnected via mitochondria-associated membranes (MAMs) supports their interrelated roles in the pathophysiology of T2DM. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance to the control of glucose homoeostasis, are still unknown. This review evaluates the involvement of mitochondria and ER independently in the development of peripheral insulin resistance, as well as their potential roles in the disruption of organelle crosstalk at MAM interfaces in the alteration of insulin signalling. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans.
Cho, J H; Bandyopadhyay, J; Lee, J; Park, C S; Ahnn, J
SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.
Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration.
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Glenn P Lobo
Full Text Available Inherited retinal disorders (IRDs result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP and Leber congenital amaurosis (LCA. TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.
Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.
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Carlos A Labriola
Full Text Available For most secretory pathway proteins, crossing the endoplasmic reticulum (ER membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD, is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C, since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.
Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.
Boussabbeh, Manel; Ben Salem, Intidhar; Prola, Alexandre; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe
Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For Permissions, please e-mail: firstname.lastname@example.com.
A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection.
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Mengding Qian
Full Text Available The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism.
Ashrafuzzaman, Mohammad
Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.
The human selenoprotein VCP-interacting membrane protein (VIMP) is non-globular and harbors a reductase function in an intrinsically disordered region
DEFF Research Database (Denmark)
Christensen, Lea Cecilie; Jensen, Njal Winther; Lages Lino Vala, Andrea
The human selenoprotein VIMP (VCP-interacting membrane protein)/SelS (selenoprotein S) localizes to the endoplasmic reticulum (ER) membrane and is involved in the process of ER-associated degradation (ERAD). To date, little is known about the presumed redox activity of VIMP, its structure and how...
Crucial role for endoplasmic reticulum stress during megakaryocyte maturation.
Lopez, Jose J; Palazzo, Alberta; Chaabane, Chiraz; Albarran, Letizia; Polidano, Evelyne; Lebozec, Kristell; Dally, Saoussen; Nurden, Paquita; Enouf, Jocelyne; Debili, Najet; Bobe, Régis
Apoptotic-like phase is an essential step for the platelet formation from megakaryocytes. How controlled is this signaling pathway remained poorly understood. The aim of this study was to determine whether endoplasmic reticulum (ER) stress-induced apoptosis occurs during thrombopoiesis. Investigation of ER stress and maturation markers in different models of human thrombopoiesis (CHRF, DAMI, MEG-01 cell lines, and hematopoietic stem cells: CD34(+)) as well as in immature pathological platelets clearly indicated that ER stress occurs transiently during thrombopoiesis. Direct ER stress induction by tunicamycin, an inhibitor of N-glycosylation, or by sarco/endoplasmic reticulum Ca(2+) ATPase type 3b overexpression, which interferes with reticular calcium, leads to some degree of maturation in megakaryocytic cell lines. On the contrary, exposure to salubrinal, a phosphatase inhibitor that prevents eukaryotic translation initiation factor 2α-P dephosphorylation and inhibits ER stress-induced apoptosis, decreased both expression of maturation markers in MEG-01 and CD34(+) cells as well as numbers of mature megakaryocytes and proplatelet formation in cultured CD34(+) cells. Taken as a whole, our research suggests that transient ER stress activation triggers the apoptotic-like phase of the thrombopoiesis process.
Ghrelin Ameliorates Asthma by Inhibiting Endoplasmic Reticulum Stress.
Fu, Tian; Wang, Lei; Zeng, Qingdi; Zhang, Yan; Sheng, Baowei; Han, Liping
This study aimed to confirm the ameliorative effect of ghrelin on asthma and investigate its mechanism. The murine model of asthma was induced by ovalbumin (OVA) treatment and assessed by histological pathology and airway responsiveness to methacholine. The total and differential leukocytes were counted. Tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 levels in bronchoalveolar lavage fluid were quantified by commercial kits. The protein levels in pulmonary tissues were measured by Western blot analysis. Ghrelin ameliorated the histological pathology and airway hyperresponsiveness in the OVA-induced asthmatic mouse model. Consistently, OVA-increased total and differential leukocytes and levels of tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 in bronchoalveolar lavage fluid were significantly attenuated by ghrelin. Ghrelin prevented the increased protein levels of the endoplasmic reticulum stress markers glucose regulated protein 78 and CCAAT/enhancer binding protein homologous protein and reversed the reduced levels of p-Akt in asthmatic mice. Ghrelin might prevent endoplasmic reticulum stress activation by stimulating the Akt signaling pathway, which attenuated inflammation and ameliorated asthma in mice. Ghrelin might be a new target for asthma therapy. Copyright © 2017. Published by Elsevier Inc.
PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes
De Matteis, MA; Luna, A; Di Tullio, G; Corda, D; Kok, JW; Luini, A; Egea, G
We reported that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholinol-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al,, 1998, J. Cell Biol. 142, 25-38), We now
Protrudin Regulates Endoplasmic Reticulum Morphology and Function Associated with the Pathogenesis of Hereditary Spastic Paraplegia*
Hashimoto, Yutaka; Shirane, Michiko; Matsuzaki, Fumiko; Saita, Shotaro; Ohnishi, Takafumi; Nakayama, Keiichi I.
Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP. PMID:24668814
Protrudin regulates endoplasmic reticulum morphology and function associated with the pathogenesis of hereditary spastic paraplegia.
Hashimoto, Yutaka; Shirane, Michiko; Matsuzaki, Fumiko; Saita, Shotaro; Ohnishi, Takafumi; Nakayama, Keiichi I
Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.
STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC
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Garbarino, J.; Pan, M. H.; Chin, H. F.
small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...
Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.
Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V
Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.
The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease
AWARD NUMBER: W81XWH-14-1-0203 TITLE: The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease PRINCIPAL...1 July 2015- 30 June 2016 4. TITLE AND SUBTITLE The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease 5a... chronic kidney disease , diabetic nephropathy, calreticulin, TGF-beta, ER stress, ultrasound, tubulointerstitial fibrosis 5 3. ACCOMPLISHMENTS a. WHAT
Role of endoplasmic reticulum stress signalling in diabetic endothelial dysfunction and atherosclerosis.
Dong, Yunzhou; Fernandes, Conrad; Liu, Yanjun; Wu, Yong; Wu, Hao; Brophy, Megan L; Deng, Lin; Song, Kai; Wen, Aiyun; Wong, Scott; Yan, Daoguang; Towner, Rheal; Chen, Hong
It is well established that diabetes mellitus accelerates atherosclerotic vascular disease. Endothelial injury has been proposed to be the initial event in the pathogenesis of atherosclerosis. Endothelium not only acts as a semi-selective barrier but also serves physiological and metabolic functions. Diabetes or high glucose in circulation triggers a series of intracellular responses and organ damage such as endothelial dysfunction and apoptosis. One such response is high glucose-induced chronic endoplasmic reticulum stress in the endothelium. The unfolded protein response is an acute reaction that enables cells to overcome endoplasmic reticulum stress. However, when chronically persistent, endoplasmic reticulum stress response could ultimately lead to endothelial dysfunction and atherosclerosis. Herein, we discuss the scientific advances in understanding endoplasmic reticulum stress-induced endothelial dysfunction, the pathogenesis of diabetes-accelerated atherosclerosis and endoplasmic reticulum stress as a potential target in therapies for diabetic atherosclerosis. © The Author(s) 2016.
Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.
Montague, Karli; Malik, Bilal; Gray, Anna L; La Spada, Albert R; Hanna, Michael G; Szabadkai, Gyorgy; Greensmith, Linda
Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain.
IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole.
Stanhope, Rebecca; Flora, Elizabeth; Bayne, Charlie; Derré, Isabelle
Membrane contact sites (MCS) are zones of contact between the membranes of two organelles. At MCS, specific proteins tether the organelles in close proximity and mediate the nonvesicular trafficking of lipids and ions between the two organelles. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of MCS involved in both tethering and lipid transfer by binding directly to proteins containing a FFAT [two phenylalanines (FF) in an acidic tract (AT)] motif. In addition to maintaining cell homeostasis, MCS formation recently emerged as a mechanism by which intracellular pathogens hijack cellular resources and establish their replication niche. Here, we investigated the mechanism by which the Chlamydia-containing vacuole, termed the inclusion, establishes direct contact with the ER. We show that the Chlamydia protein IncV, which is inserted into the inclusion membrane, displays one canonical and one noncanonical FFAT motif that cooperatively mediated the interaction of IncV with VAP. IncV overexpression was sufficient to bring the ER in close proximity of IncV-containing membranes. Although IncV deletion partially decreased VAP association with the inclusion, it did not suppress the formation of ER-inclusion MCS, suggesting the existence of redundant mechanisms in MCS formation. We propose a model in which IncV acts as one of the primary tethers that contribute to the formation of ER-inclusion MCS. Our results highlight a previously unidentified mechanism of bacterial pathogenesis and support the notion that cooperation of two FFAT motifs may be a common feature of VAP-mediated MCS formation. Chlamydia-host cell interaction therefore constitutes a unique system to decipher the molecular mechanisms underlying MCS formation. Published under the PNAS license.
The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate-washed membrane standard.
Peng, Lifeng; Kapp, Eugene A; Fenyö, David; Kwon, Min-Seok; Jiang, Pu; Wu, Songfeng; Jiang, Ying; Aguilar, Marie-Isabel; Ahmed, Nikhat; Baker, Mark S; Cai, Zongwei; Chen, Yu-Ju; Van Chi, Phan; Chung, Maxey C M; He, Fuchu; Len, Alice C L; Liao, Pao-Chi; Nakamura, Kazuyuki; Ngai, Sai Ming; Paik, Young-Ki; Pan, Tai-Long; Poon, Terence C W; Salekdeh, Ghasem Hosseini; Simpson, Richard J; Sirdeshmukh, Ravi; Srisomsap, Chantragan; Svasti, Jisnuson; Tyan, Yu-Chang; Dreyer, Florian S; McLauchlan, Danyl; Rawson, Pisana; Jordan, T William
The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Kulprathipanja, Santi; Kulkarni, Sudhir S.; Funk, Edward W.
A multicomponent membrane which may be used for separating various components which are present in a fluid feed mixture comprises a mixture of a plasticizer such as a glycol and an organic polymer cast upon a porous organic polymer support. The membrane may be prepared by casting an emulsion or a solution of the plasticizer and polymer on the porous support, evaporating the solvent and recovering the membrane after curing.
Induction of Apoptosis by Hypertension Via Endoplasmic Reticulum Stress
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Yingying Sun
Full Text Available Background/Aims: Endoplasmic reticulum (ER stress is one of the intrinsic apoptosis pathways, and cardiac apoptosis can occur in cardiovascular diseases, such as hypertension. However, the mechanisms by which ER stress leads to apoptosis remain enigmatic, particularly in the progression from cardiac hypertrophy to diastolic heart failure due to hypertension. Methods: We used spontaneously hypertensive rats (SHRs to investigate possible signalling pathways for ER stress. Results: We found that cardiac protein and mRNA levels of glucose-regulated protein 78 were up-regulated. In addition, the CHOP- and caspase-12-dependent pathways, but not that of JNK, were activated in the SHR rats. Conclusions: These results suggest that ER stress can contribute to myocardial apoptosis during hypertensive disease.
How Are Proteins Reduced in the Endoplasmic Reticulum?
Ellgaard, Lars; Sevier, Carolyn S; Bulleid, Neil J
The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Endoplasmic Reticulum Stress-Related Factors Protect against Diabetic Retinopathy
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Wei-Kun Hu
Full Text Available The endoplasmic reticulum (ER is a principal mediator of signal transduction in the cell, and disruption of its normal function (a mechanism known as ER stress has been associated with the pathogenesis of several diseases. ER stress has been demonstrated to contribute to onset and progression of diabetic retinopathy (DR by induction of multiple inflammatory signaling pathways. Recent studies have begun to describe the gene expression profile of ER stress-related genes in DR; moreover, genes that play a protective role against DR have been identified. P58IPK was determined to be able to reduce retinal vascular leakage under high glucose conditions, thus protecting retinal cells. It has also been found by our lab that ER-associated protein degradation factors exhibit significantly different expression patterns in rat retinas under sustained high glucose conditions. Future research based upon these collective genomic findings will contribute to our overall understanding of DR pathogenesis as well as identify potential therapeutic targets.
Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin
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Craig Myrum
Full Text Available Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF, Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG. After induction of long-term potentiation (LTP in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL. Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.
Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells
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Hagen, Christoph, E-mail: email@example.com [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN (United Kingdom); Werner, Stephan, E-mail: email@example.com [Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Wilhelm-Conrad-Röntgen Campus, 12489 Berlin (Germany); Carregal-Romero, Susana, E-mail: firstname.lastname@example.com [Fachbereich Physik, Philipps Universität Marburg, Marburg 35043 (Germany); Malhas, Ashraf N., E-mail: firstname.lastname@example.com [Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE (United Kingdom); Klupp, Barbara G., E-mail: email@example.com [Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems (Germany); Guttmann, Peter, E-mail: email@example.com [Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Wilhelm-Conrad-Röntgen Campus, 12489 Berlin (Germany); Rehbein, Stefan, E-mail: firstname.lastname@example.com [Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Wilhelm-Conrad-Röntgen Campus, 12489 Berlin (Germany); Henzler, Katja, E-mail: email@example.com [Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Wilhelm-Conrad-Röntgen Campus, 12489 Berlin (Germany); Mettenleiter, Thomas C., E-mail: email@example.com [Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems (Germany); and others
Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. - Highlights: • Nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated cells. • New polyelectrolyte-Qdot{sup ®} 605 coated gold beads were employed as fiducials. • Saquinavir can induce a strong apoptotic phenotype in the nucleus. • CryoXT is an auspicious imaging technique in apoptosis research.
Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum
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Thea Bismo Strøm
Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.
The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum.
Truchan, Hilary K; Cockburn, Chelsea L; Hebert, Kathryn S; Magunda, Forgivemore; Noh, Susan M; Carlyon, Jason A
The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly
Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells.
Zhang, Xin-Yu; Yang, Shu-Meng; Zhang, Hao-Peng; Yang, Yue; Sun, Shi-Bo; Chang, Jian-Ping; Tao, Xuan-Chen; Yang, Tuo-Yun; Liu, Chun; Yang, Yan-Mei
Arsenic trioxide has been proven to trigger apoptosis in human hepatocellular carcinoma cells. Endoplasmic reticulum stress has been known to be involved in apoptosis through the induction of CCAAT/enhancer-binding protein homologous protein. However, it is unknown whether endoplasmic reticulum stress mediates arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells. Our data showed that arsenic trioxide significantly induced apoptosis in human hepatocellular carcinoma cells. Furthermore, arsenic trioxide triggered endoplasmic reticulum stress, as indicated by endoplasmic reticulum dilation, upregulation of glucose-regulated protein 78 and CCAAT/enhancer-binding protein homologous protein. We further found that 4-phenylbutyric acid, an inhibitor of endoplasmic reticulum stress, alleviated arsenic trioxide-induced expression of CCAAT/enhancer-binding protein homologous protein. More important, knockdown of CCAAT/enhancer-binding protein homologous protein by siRNA or inhibition of endoplasmic reticulum stress by 4-phenylbutyric acid alleviated apoptosis induced by arsenic trioxide. Consequently, our results suggested that arsenic trioxide could induce endoplasmic reticulum stress-mediated apoptosis in hepatocellular carcinoma cells, and that CCAAT/enhancer-binding protein homologous protein might play an important role in this process. Copyright © 2015 Elsevier Ltd. All rights reserved.
Staszak, Katarzyna
The membrane processes have played important role in the industrial separation process. These technologies can be found in all industrial areas such as food, beverages, metallurgy, pulp and paper, textile, pharmaceutical, automotive, biotechnology and chemical industry, as well as in water treatment for domestic and industrial application. Although these processes are known since twentieth century, there are still many studies that focus on the testing of new membranes' materials and determining of conditions for optimal selectivity, i. e. the optimum transmembrane pressure (TMP) or permeate flux to minimize fouling. Moreover the researchers proposed some calculation methods to predict the membrane processes properties. In this article, the laboratory scale experiments of membrane separation techniques, as well their validation by calculation methods are presented. Because membrane is the "heart" of the process, experimental and computational methods for its characterization are also described.
Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: a protective role.
Cheng, Yu-Chi; Chang, Jer-Ming; Chen, Chien-An; Chen, Hung-Chun
Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage. © 2014 by the Society for Experimental Biology and Medicine.
Connecting membranes to the actin cytoskeleton.
Wang, Pengwei; Hawkins, Tim J; Hussey, Patrick J
In plants, the actin cytoskeleton plays a major role in organelle movement, cargo transport, maintaining cell polarity and controlling the morphogenesis of endomembrane systems. All of these events require a direct connection between membrane structures and the cytoskeleton. Our knowledge in this field has been greatly advanced by a few recent discoveries including the identification of the plant specific NETWORKED family of proteins, which can mediate such linkages. Other proteins that are known to regulate actin nucleation and polymerization are also likely to be involved, but many key questions still remain unanswered. In this paper, we will focus on recent research on the interfaces between the actin cytoskeleton and membranes of the endoplasmic reticulum, the vacuole and autophagosomes. Copyright © 2017 Elsevier Ltd. All rights reserved.
Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway.
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José Peña
Full Text Available The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV to study the role of the unfolded protein response (UPR and sterol-regulatory-element-binding-protein-2 (SREBP-2 pathway in the rearrangement and expansion of the endoplasmic reticulum (ER early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.
Hepler, P.
Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.
Role and mechanism of endoplasmic reticulum stress and Ca2+ overload in pulmonary endothelial cell damage induced by heat stress
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Bao-jun YU
Full Text Available Objective To observe the effect of different temperatures on endoplasmic reticulum stress, calcium overload, mitochondria and cell damage in pulmonary microvascular endothelial cells (PMVEC induced by heat stress, and clarify the mechanism of endothelial cell injury in the process of heat stress to provide experimental basis for clinical prevention and treatment of heat stree. Methods Heat stress model of PMVEC cell was set up. Control group cells were incubated at 37℃, 5%CO2, while heat stress group cells were incubated at 39℃, 41℃, 43℃ for 2h, respectively, then further incubated at 37℃, 5%CO2 for 6h. Pretreatment of cells with 20μmol/L BAPTA-AM or 50μmol/L CsA before heat stress at 43℃. The protein levels of p-PERK, PERK p-eIF2a, eIF2a, ATF4 and GRP78 were analyzed by Western blotting. Intracellular Ca2+, mitochondrial membrane potential and the changes in mitochondrial permeability transition pore were investigated by flow cytometry. The change of caspase-3 was detected by Caspase Assay Kit. Millicell-ERS Volt-Ohm Meter and Accessories was used for determining the changes of transepithelium electrical resistance (TER. Results Compared with the control group, with the increase of heat stress temperature (41-43℃, the phosphorylation of p-PERK and p-eIF2a protein and the expressions of ATF4 and GRP78 proteins were gradually activated, intracellular Ca2+ increased, MPTP pore was opened, mitochondrial membrane potential decreased, cell permeability increased and apoptosis occurred, and it was the most obvious in the 43℃ heat stress group, and the difference was statistically significant (P<0.05. Pretreatment with Ca2+ inhibitors promoted the recovery of the MPTP hole, mitochondrial membrane potential and cell permeability, and reduced the occurrence of apoptosis. While pretreatment with the mitochondrial protective agent did not reduce the release of Ca2+, but it could promote the recovery of cell permeability and reduce the
DEFF Research Database (Denmark)
Hanczyc, Martin M; Monnard, Pierre-Alain
Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during...... the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions...
The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.
Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Chen, Jianping
Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. © 2013 BSPP AND JOHN WILEY & SONS LTD.
Endoplasmic reticulum-resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica.
Hanadate, Yuki; Saito-Nakano, Yumiko; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi
Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans-Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down-regulation of EhRab8A by small antisense RNA-mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS-PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene-silenced strain. Further, overexpression of wild-type EhRab8A augmented phagocytosis, whereas expression of the dominant-negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER-resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.
Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.
Riaz Ahmed, Kausar Begam; Kanduluru, Ananda Kumar; Feng, Li; Fuchs, Philip L; Huang, Peng
Metastatic melanoma is the most aggressive of all skin cancers and is associated with poor prognosis owing to lack of effective treatments. 25-epi Ritterostatin GN1N is a novel antitumor agent with yet undefined mechanisms of action. We sought to delineate the antitumor mechanisms of 25-epi Ritterostatin GN1N in melanoma cells to determine the potential of this compound as a treatment for melanoma. Activation of the endoplasmic reticulum (ER) stress protein glucose-regulated protein 78 (GRP78) has been associated with increased melanoma progression, oncogenic signaling, drug resistance, and suppression of cell death. We found that 25-epi Ritterostatin GN1N induced cell death in melanoma cells at nanomolar concentrations, and this cell death was characterized by inhibition of GRP78 expression, increased expression of the ER stress marker CHOP, loss of mitochondrial membrane potential, and lipidation of the autophagy marker protein LC3B. Importantly, normal melanocytes exhibited limited sensitivity to 25-epi Ritterostatin GN1N. Subsequent in vivo results demonstrated that 25-epi Ritterostatin GN1N reduced melanoma growth in mouse tumor xenografts and did not affect body weight, suggesting minimal toxicity. In summary, our findings indicate that 25-epi Ritterostatin GN1N causes ER stress and massive autophagy, leading to collapse of mitochondrial membrane potential and cell death in melanoma cells, with minimal effects in normal melanocytes. Thus, 25-epi Ritterostatin GN1N is a promising anticancer agent that warrants further investigation.
DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress
Yuste-Checa, Patricia; Vega, Ana I.; Martín-Higueras, Cristina; Medrano, Celia; Gámez, Alejandra; Desviat, Lourdes R.; Ugarte, Magdalena
Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij), and limb-girdle congenital myasthenic syndrome (CMS) with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT), the protein encoded by DPAGT1, is an endoplasmic reticulum (ER)-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met), c.791T>G (p.Val264Gly), c.901C>T (p.Arg301Cys), c.902G>A (p.Arg301His), c.1154T>G (p.Leu385Arg), and of the novel mutation c.329T>C (p.Phe110Ser), were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress) was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies. PMID:28662078
DPAGT1-CDG: Functional analysis of disease-causing pathogenic mutations and role of endoplasmic reticulum stress.
Directory of Open Access Journals (Sweden)
Patricia Yuste-Checa
Full Text Available Pathogenic mutations in DPAGT1 are manifested as two possible phenotypes: congenital disorder of glycosylation DPAGT1-CDG (also known as CDG-Ij, and limb-girdle congenital myasthenic syndrome (CMS with tubular aggregates. UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosamine phosphotransferase (GPT, the protein encoded by DPAGT1, is an endoplasmic reticulum (ER-resident protein involved in an initial step in the N-glycosylation pathway. The aim of the present study was to examine the effect of six variants in DPAGT1 detected in patients with DPAGT1-CDG, and the role of endoplasmic reticulum stress, as part of the search for therapeutic strategies to use against DPAGT1-CDG. The effect of the six mutations, i.e., c.358C>A (p.Leu120Met, c.791T>G (p.Val264Gly, c.901C>T (p.Arg301Cys, c.902G>A (p.Arg301His, c.1154T>G (p.Leu385Arg, and of the novel mutation c.329T>C (p.Phe110Ser, were examined via the analysis of DPAGT1 transcriptional profiles and GTP levels in patient-derived fibroblasts. In addition, the transient expression of different mutations was analysed in COS-7 cells. The results obtained, together with those of bioinformatic studies, revealed these mutations to affect the splicing process, the stability of GTP, or the ability of this protein to correctly localise in the ER membrane. The unfolded protein response (UPR; the response to ER stress was found not to be active in patient-derived fibroblasts, unlike that seen in cells from patients with PMM2-CDG or DPM1-CDG. Even so, the fibroblasts of patients with DPAGT1-CDG seemed to be more sensitive to the stressor tunicamycin. The present work improves our knowledge of DPAGT1-CDG and provides bases for developing tailored splicing and folding therapies.
Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil
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Héctor M Mora-Montes
Full Text Available Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.
Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.
Sainath, Rajiv; Armijo-Weingart, Lorena; Ketscheck, Andrea; Xu, Zhuxuan; Li, Shuxin; Gallo, Gianluca
Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150 Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017. © 2017 Wiley Periodicals, Inc.
Annatto prevents retinal degeneration induced by endoplasmic reticulum stress in vitro and in vivo.
Tsuruma, Kazuhiro; Shimazaki, Hiroki; Nakashima, Ken-Ichi; Yamauchi, Mika; Sugitani, Sou; Shimazawa, Masamitsu; Iinuma, Munekazu; Hara, Hideaki
Annatto (Bixa orellana) seeds have been used as a colorant in butter and in a variety of other foods. In this study, we investigated the amelioration of retinal damage by an acetone extract of annatto (A-ext.), bixin (a main component of annatto), and four bixin derivatives (Bx-1, Bx-2, Bx-3, and Bx-4) that we have synthesized. We used cultured retinal ganglion cells (RGC-5) to examine in vitro effects of A-ext. on stress pathways, focusing on intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, caspase-3 activation, and cell membrane damage. In vivo retinal damage in mice following intravitreous injection of tunicamycin was evaluated by counting the cell numbers in the ganglion cell layer (GCL) and measuring the thickness of outer nuclear layer (ONL). A-ext., bixin, and Bx-1 treatment inhibited both tunicamycin- and H₂O₂-induced cell death. Bixin derivatives also inhibited tunicamycin-induced cell death. Treatment with A-ext., bixin, and Bx-1 reduced tunicamycin-induced caspase-3 activity and inhibited the inversion of phosphatidylserine, an early apoptotic event without antioxidant effect or reduction of ER stress itself. A-ext., bixin, and Bx-1 significantly inhibited the tunicamycin-induced loss of cells from the GCL, and these materials also suppressed the tunicamycin-induced thinning of ONL. A-ext., its main component bixin, and bixin derivatives may therefore be useful for preventive and therapeutic treatment of retinal-related diseases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves
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Joseph Minu
Full Text Available Abstract Background The N-terminal proline-rich domain (Zera of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER-derived protein bodies (PBs when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. Results We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed. Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. Conclusions This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of
The flexibility and dynamics of the tubules in the endoplasmic reticulum.
Georgiades, Pantelis; Allan, Victoria J; Wright, Graham D; Woodman, Philip G; Udommai, Parinya; Chung, Manloeng A; Waigh, Thomas A
The endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 μm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 μm, and provides the tubule's persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.
Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase.
Zhang, Z; Chen, D; Wheatly, M G
The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of
Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast
DEFF Research Database (Denmark)
Busti, Stefano; Mapelli, Valeria; Tripodi, Farida
Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully...
Toll-like receptor 4-induced endoplasmic reticulum stress contributes to endothelial dysfunction
Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Endoplasmic reticulum (ER) stress is implicated as one of the mechanisms for pathophysiology of various cardiometabolic syndromes, including insulin resistance and endothelial dysfunction. ...
Placental endoplasmic reticulum stress and acidosis: relevant aspects in gestational diabetes.
Jawerbaum, Alicia
In this issue, Yung and colleagues (doi: 10.1007/s00125-016-4040-2 ) report endoplasmic reticulum stress in the placenta of patients with gestational diabetes mellitus. With the use of a trophoblast-like cell line, these authors identify putative mechanisms involved in, and treatments to prevent the induction of endoplasmic reticulum stress. Here, the relevance and possible implications of these findings and areas for further research are discussed.
Analysis of endoplasmic reticulum stress in placentas of HIV-infected women treated with protease inhibitors.
Brüning, Ansgar; Kimmich, Tanja; Brem, German J; Buchholtz, Marie L; Mylonas, Ioannis; Kost, Bernd; Weizsäcker, Katharina; Gingelmaier, Andrea
Combined antiretroviral therapy has proven efficacy in decreasing vertical HIV transmission. However, endoplasmic reticulum stress is a known side effect of HIV protease inhibitors. We investigated endoplasmic reticulum stress in placentas of HIV-infected and uninfected mothers by PCR-based splicing analysis of the specific endoplasmic reticulum stress marker XBP1 in post-delivery placental samples of uninfected mothers and in HIV-infected mothers taking antiretroviral therapy. No elevated XBP1 splicing could be detected in placentas of uninfected mothers and most of the mothers receiving combined anti-retroviral therapy. However, markedly elevated XBP1 splicing was found in the placentas of three individuals on combined antiviral therapy, all receiving lopinavir or atazanavir. In vitro experiments confirmed induction of endoplasmic reticulum stress by lopinavir and atazanavir in trophoblast-derived cell lines. Since endoplasmic reticulum stress occurred in selective patients only, individual differences in susceptibility of HIV-infected mothers to protease inhibitor induced endoplasmic reticulum stress can be postulated. Copyright © 2014 Elsevier Inc. All rights reserved.
Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology.
Chong, Wai Chin; Shastri, Madhur D; Eri, Rajaraman
The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases.
Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes.
Hassan, Hossam; Tian, Xuefei; Inoue, Kazunori; Chai, Nathan; Liu, Chang; Soda, Keita; Moeckel, Gilbert; Tufro, Alda; Lee, Ann-Hwee; Somlo, Stefan; Fedeles, Sorin; Ishibe, Shuta
Podocytes are terminally differentiated epithelial cells that reside along the glomerular filtration barrier. Evidence suggests that after podocyte injury, endoplasmic reticulum stress response is activated, but the molecular mechanisms involved are incompletely defined. In a mouse model, we confirmed that podocyte injury induces endoplasmic reticulum stress response and upregulated unfolded protein response pathways, which have been shown to mitigate damage by preventing the accumulation of misfolded proteins in the endoplasmic reticulum. Furthermore, simultaneous podocyte-specific genetic inactivation of X-box binding protein-1 (Xbp1), a transcription factor activated during endoplasmic reticulum stress and critically involved in the untranslated protein response, and Sec63, a heat shock protein-40 chaperone required for protein folding in the endoplasmic reticulum, resulted in progressive albuminuria, foot process effacement, and histology consistent with ESRD. Finally, loss of both Sec63 and Xbp1 induced apoptosis in podocytes, which associated with activation of the JNK pathway. Collectively, our results indicate that an intact Xbp1 pathway operating to mitigate stress in the endoplasmic reticulum is essential for the maintenance of a normal glomerular filtration barrier. Copyright © 2016 by the American Society of Nephrology.
[Research on olaquindox induced endoplasmic reticulum stress related apoptosis on nephrotoxicity].
Li, Zhongsheng; Yu, Changyan; Chen, Xiao; Zhang, Bin; Cao, Peng; Li, Bin; Xiao, Jingwei
Renal tubular epithelial cell were exposed to olaquindox and detected the ROS and apoptosis related proteins, to investigate the renal tubular epithelial cell apoptosis through endoplasmic reticulum stress mediated pathway induced by olaquindox. MTT assay (1, 2, 3, 4, 5, 6, 7and 8 μmol/ml olaquindox exposure) was used to detect the effects of olaquindox on renal tubular epithelial cell proliferation to determine test concentrations. Hoechst-33258 was used to detect morphological changes on apoptotic cells in each group. Flow cytometry method was applied to detect the apoptosis rate and intracellular reactive oxygen, and western blot assay was performed to detect the levels of endoplasmic reticulum stress-related apoptosis proteins, GRP78, GRP94 and CHOP. According to results of the MTT test, 1, 2, 3 and 4 μmol/ml olaquindox concentrations were determined for apoptosis analysis. With the increase of olaquindox concentration, apoptosis rate and levels of endoplasmic reticulum stress related apoptosis pathway protein GRP78, GRP94 and CHOP increased, levels of ROS were increased in every groups (P endoplasmic reticulum stress related apoptosis pathway protein GRP78 and GRP94 increased in 12 and 24 h olaquindox exposure groups, whereas in groups of olaquindox exposed for 6, 12 and 24 h, levels of ROS and endoplasmic reticulum stress related apoptosis pathway protein CHOP increased (P endoplasmic reticulum stress-related apoptosis maybe the associated toxicity pathway.
Estrogen protects SGC7901 cells from endoplasmic reticulum stress-induced apoptosis by the Akt pathway
Several previous studies have demonstrated that estrogen may protect cancer cells from endoplasmic reticulum stress-induced apoptosis. However, the molecular mechanisms involved are not fully understood. In the present study, human gastric adenocarcinoma SGC7901 cells were treated with tunicamycin (TM) to induce endoplasmic reticulum stress. This was demonstrated by increased glucose-regulated protein 78 expression and enhanced phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase. Endoplasmic reticulum stress induced caspase-3-mediated apoptosis with the inhibition of Akt; the latter of which was measured by the activity-dependent phosphorylation at Ser473 of Akt. Simultaneous treatment of 10−9 M 17β-estradiol (E2) with TM may protect SGC7901 cells from endoplasmic reticulum stress-induced apoptosis by counteracting the inhibitory effect of TM on Akt, causing an increase in the phosphorylation of Ser473-Akt. It was concluded that low concentrations of E2 may counteract endoplasmic reticulum stress-induced inactivation of Akt to block caspase-3-mediated apoptosis. PMID:24396487
Tanjore, Harikrishna; Lawson, William E.; Blackwell, Timothy S.
Current evidence suggests a prominent role for endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in fibrotic conditions affecting a number of internal organs, including the lungs, liver, GI tract, kidney, and heart. ER stress enhances the susceptibility of structural cells, in most cases the epithelium, to pro-fibrotic stimuli. Studies suggest that ER stress facilitates fibrotic remodeling through activation of pro-apoptotic pathways, induction of epithelial-mesenchymal transition, and promotion of inflammatory responses. While genetic mutations that lead to ER stress underlie some cases of fibrosis, including lung fibrosis secondary to mutations in surfactant protein C (SFTPC), a variety of other factors can cause ER stress. These ER stress inducing factors include metabolic abnormalities, oxidative stress, viruses, and environmental exposures. Interestingly, the ability of the ER to maintain homeostasis under stress diminishes with age, potentially contributing to the fact that fibrotic disorders increase in incidence with aging. Taken together, underlying ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in different forms of tissue fibrosis. Further work is needed to better define the mechanisms by which ER stress facilitates progressive tissue fibrosis. In addition, it remains to be seen whether targeting ER stress and the UPR could have therapeutic benefit. PMID:23201247
The Role of Endoplasmic Reticulum Stress in Human Pathology
Oakes, Scott A.; Papa, Feroz R.
Numerous genetic and environmental insults impede the ability of cells to properly fold and posttranslationally modify secretory and transmembrane proteins in the endoplasmic reticulum (ER), leading to a buildup of misfolded proteins in this organelle—a condition called ER stress. ER-stressed cells must rapidly restore protein-folding capacity to match protein-folding demand if they are to survive. In the presence of high levels of misfolded proteins in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) induces a set of transcriptional and translational events that restore ER homeostasis. However, if ER stress persists chronically at high levels, a terminal UPR program ensures that cells commit to self-destruction. Chronic ER stress and defects in UPR signaling are emerging as key contributors to a growing list of human diseases, including diabetes, neurodegeneration, and cancer. Hence, there is much interest in targeting components of the UPR as a therapeutic strategy to combat these ER stress–associated pathologies. PMID:25387057
The endoplasmic reticulum stress response and diabetic kidney disease
Sharma, Kumar
The endoplasmic reticulum (ER) folds and modifies proteins; however, during conditions of cellular stress, unfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR, also referred to as the ER stress response, activates three distinct signaling cascades that are designed to globally reduce transcription and translation. The three major arms of the mammalian UPR include 1) protein kinase RNA (PKR)-like ER kinase (PERK), 2) inositol-requiring protein-1 (IRE1α), and 3) activating transcription factor-6 (ATF6) pathways. The PERK pathway rapidly attenuates protein translation, whereas the ATF6 and IRE1α cascades transcriptionally upregulate ER chaperone genes that promote proper folding and ER-associated degradation (ERAD) of proteins. This integrated response in turn allows the folding machinery of the ER to catch up with the backlog of unfolded proteins. The ER stress response plays a role in a number of pathophysiological processes, including pancreatic β-cell failure and apoptosis. The goals of the current review are to familiarize investigators with cellular and tissue activation of this response in the rodent and human diabetic kidney. Additionally, we will review therapeutic modulators of the ER stress response and discuss their efficacy in models of diabetic kidney disease. The ER stress response has both protective and deleterious features. A better understanding of the molecular pathways regulated during this process in a cell- and disease-specific manner could reveal novel therapeutic strategies in chronic renal diseases, including diabetic kidney disease. PMID:21345978
Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate
Corazzari, Marco; Gagliardi, Mara; Fimia, Gian Maria; Piacentini, Mauro
Perturbation of endoplasmic reticulum (ER) homeostasis results in a stress condition termed "ER stress" determining the activation of a finely regulated program defined as unfolded protein response (UPR) and whose primary aim is to restore this organelle's physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s) regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch. PMID:28491820
Role of endoplasmic reticulum stress in endothelial dysfunction.
Cimellaro, A; Perticone, M; Fiorentino, T V; Sciacqua, A; Hribal, M L
Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of several human disorders, including cardiovascular disease (CVD). CVD recognizes endothelial dysfunction (ED) as its pathogenetic primum movens; interestingly a large body of evidence has identified the unchecked ER stress response as a main actor in vascular damage elicited by various cardio-metabolic risk factors. In the present Review, we summarize findings from experimental studies on the ER stress-related ED, focusing on the mechanisms underlying this association. Different noxious agents, such as hyperhomocysteinemia, hyperlipidemia, hyperglycemia and chronic inflammation, induce ED promoting an amplified ER stress response as demonstrated by several studies in animal models, as well as in human primary and immortalized endothelial cells. ER stress represents therefore a key mediator of vascular damage, operating in a setting of increased inflammatory burden and oxidative stress, thus contributing to foster a vicious pathogenic cycle. Experimental studies summarized in this Review strongly suggest that an unchecked ER stress response plays a central role in the pathogenesis of ED and, consequently, CVD. Counteracting ER stress may thus represent a promising, even if largely unexplored as-yet, therapeutic approach aimed to prevent vascular damage, slowing the progression from ED to cardiovascular events. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.
Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate
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Marco Corazzari
Full Text Available Perturbation of endoplasmic reticulum (ER homeostasis results in a stress condition termed "ER stress" determining the activation of a finely regulated program defined as unfolded protein response (UPR and whose primary aim is to restore this organelle's physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch.
Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.
Xue, Yong; Schmollinger, Stefan; Attar, Narsis; Campos, Oscar A; Vogelauer, Maria; Carey, Michael F; Merchant, Sabeeha S; Kurdistani, Siavash K
The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 ( VPS13 ) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Chlorpyrifos induces endoplasmic reticulum stress in JEG-3 cells.
Reyna, Luciana; Flores-Martín, Jésica; Ridano, Magali E; Panzetta-Dutari, Graciela M; Genti-Raimondi, Susana
Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50μM or 100μM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53. Copyright © 2016 Elsevier Ltd. All rights reserved.
Proteomic analysis of endoplasmic reticulum stress responses in rice seeds.
Qian, Dandan; Tian, Lihong; Qu, Leqing
The defects in storage proteins secretion in the endosperm of transgenic rice seeds often leads to endoplasmic reticulum (ER) stress, which produces floury and shrunken seeds, but the mechanism of this response remains unclear. We used an iTRAQ-based proteomics analysis of ER-stressed rice seeds due to the endosperm-specific suppression of OsSar1 to identify changes in the protein levels in response to ER stress. ER stress changed the expression of 405 proteins in rice seed by >2.0- fold compared with the wild-type control. Of these proteins, 140 were upregulated and 265 were downregulated. The upregulated proteins were mainly involved in protein modification, transport and degradation, and the downregulated proteins were mainly involved in metabolism and stress/defense responses. A KOBAS analysis revealed that protein-processing in the ER and degradation-related proteasome were the predominant upregulated pathways in the rice endosperm in response to ER stress. Trans-Golgi protein transport was also involved in the ER stress response. Combined with bioinformatic and molecular biology analyses, our proteomic data will facilitate our understanding of the systemic responses to ER stress in rice seeds.
Heme oxygenase-1 comes back to endoplasmic reticulum
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Kim, Hong Pyo [School of Biological Sciences, Ulsan University (Korea, Republic of); Pae, Hyun-Ock [Department of Immunology, Wonkwang University School of Medicine (Korea, Republic of); Back, Sung Hun; Chung, Su Wol [School of Biological Sciences, Ulsan University (Korea, Republic of); Woo, Je Moon [Department of Opthalmology, Ulasn University Hospital (Korea, Republic of); Son, Yong [Department of Anesthesiology and Pain Medicine, Wonkwang University School of Medicine (Korea, Republic of); Chung, Hun-Taeg, E-mail: firstname.lastname@example.com [School of Biological Sciences, Ulsan University (Korea, Republic of)
Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.
Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.
Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki
Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.
Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells
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Keisuke Sato
Full Text Available Schwann cell injury is caused by diabetic neuropathy. The apoptosis of Schwann cells plays a pivotal role in diabetic nerve dysfunction. Glycolaldehyde is a precursor of advanced glycation end products that contribute to the pathogenesis of diabetic neuropathy. In this study, we examined whether glycolaldehyde induces endoplasmic reticulum (ER stress and apoptosis in rat Schwann cells. Schwann cells treated with 500 μM glycolaldehyde showed morphological changes characteristic of apoptosis. Glycolaldehyde activated apoptotic signals, such as caspase-3 and caspase-8. Furthermore, it induced ER stress response involving RNA-dependent protein kinase-like ER kinase (PERK, inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α, and eukaryotic initiation factor 2α (eIF2α. In addition, glycolaldehyde activated CCAAT/enhancer-binding homologous protein (CHOP, an ER stress response factor crucial to executing apoptosis. Knockdown of nuclear factor E2-related factor 2 (Nrf2, which is involved in the promotion of cell survival following ER stress, enhanced glycolaldehyde-induced cytotoxicity, indicating that Nrf2 plays a protective role in the cytotoxicity caused by glycolaldehyde. Taken together, these findings indicate that glycolaldehyde is capable of inducing apoptosis and ER stress in Schwann cells. The ER stress induced by glycolaldehyde may trigger the glycolaldehyde-induced apoptosis in Schwann cells. This study demonstrated for the first time that glycolaldehyde induced ER stress.
Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.
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Xue Yu
Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.
Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Diabetes.
Rocha, Milagros; Diaz-Morales, Noelia; Rovira-Llopis, Susana; Escribano-Lopez, Irene; Bañuls, Celia; Hernandez-Mijares, Antonio; Diamanti-Kandarakis, Evanthia; Victor, Victor M
It is generally accepted that mitochondrial dysfunction and endoplasmic reticulum (ER) stress are related to insulin resistance and type 2 diabetes. Mitochondria use substrates from lipid and glucose metabolism in order to generate ATP, and when mitochondrial O2 consumption is decreased due to an altered metabolism there is an increase in reactive oxygen species (ROS) that can impair different types of molecules and cells, especially in β- cells during type 2 diabetes. Furthermore, the maintenance of ER function in insulin-secreting β-cells is crucial, and when ER homeostasis is disrupted, the ER develops an unfolded protein response (UPR) in order to maintain the homeostasis of this organelle. However, when homeostasis fails in mitochondria and ER, these organelles can initiate death signalling pathways. New research has suggested that hyperlipidemia and hyperliglucaemia, known as key factors of type 2 diabetes (T2D), disrupt mitochondrial activity and ER homeostasis, thus triggering a disruption of energy metabolism, unresolvable UPR activation and β-cell death. This review explains the mechanisms of mitochondrial function and ER stress related to the pathological effects of type 2 diabetes in different tissues.
Apoptosis, autophagy & endoplasmic reticulum stress in diabetes mellitus.
Demirtas, Levent; Guclu, Aydin; Erdur, Fatih Mehmet; Akbas, Emin Murat; Ozcicek, Adalet; Onk, Didem; Turkmen, Kultigin
The prevalence of diabetes mellitus (DM) is increasing secondary to increased consumption of food and decreased physical activity worldwide. Hyperglycaemia, insulin resistance and hypertrophy of pancreatic beta cells occur in the early phase of diabetes. However, with the progression of diabetes, dysfunction and loss of beta cells occur in both types 1 and 2 DM. Programmed cell death also named apoptosis is found to be associated with diabetes, and apoptosis of beta cells might be the main mechanism of relative insulin deficiency in DM. Autophagic cell death and apoptosis are not entirely distinct programmed cell death mechanisms and share many of the regulator proteins. These processes can occur in both physiologic and pathologic conditions including DM. Besides these two important pathways, endoplasmic reticulum (ER) also acts as a cell sensor to monitor and maintain cellular homeostasis. ER stress has been found to be associated with autophagy and apoptosis. This review was aimed to describe the interactions between apoptosis, autophagy and ER stress pathways in DM.
Sphingolipids activate the endoplasmic reticulum stress surveillance pathway.
Piña, Francisco; Yagisawa, Fumi; Obara, Keisuke; Gregerson, J D; Kihara, Akio; Niwa, Maho
Proper inheritance of functional organelles is vital to cell survival. In the budding yeast, Saccharomyces cerevisiae, the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway ensures that daughter cells inherit a functional ER. Here, we show that the ERSU pathway is activated by phytosphingosine (PHS), an early biosynthetic sphingolipid. Multiple lines of evidence support this: (1) Reducing PHS levels with myriocin diminishes the ability of cells to induce ERSU phenotypes. (2) Aureobasidin A treatment, which blocks conversion of early intermediates to downstream complex sphingolipids, induces ERSU. (3) orm1Δorm2Δ cells, which up-regulate PHS, show an ERSU response even in the absence of ER stress. (4) Lipid analyses confirm that PHS levels are indeed elevated in ER-stressed cells. (5) Lastly, the addition of exogenous PHS is sufficient to induce all ERSU phenotypes. We propose that ER stress elevates PHS, which in turn activates the ERSU pathway to ensure future daughter-cell viability. © 2018 Piña et al.
Endoplasmic reticulum redox state is not perturbed by pharmacological or pathological endoplasmic reticulum stress in live pancreatic β-cells.
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Irmgard Schuiki
Full Text Available Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor and mCherry protein driven by a GRP78 promoter (UPR-sensor. Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.
Endoplasmic reticulum stress affects the transport of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum in S.cerevisiae.
Kannan, Muthukumar; Sivaprakasam, Chinnarasu; Prinz, William A; Nachiappan, Vasanthi
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two of the most abundant phospholipids in cells. Although both lipids can be synthesized in the endoplasmic reticulum (ER), in S. cerevisiae PE can also be produced in mitochondria and endosomes; this PE can be transported back to the ER where it is converted to PC. In this study we found that dithiothreitol (DTT), which induces ER stress, decreases PE export from mitochondria to the ER. This results in decreased levels of total cellular PC and mitochondrial PC. These decreases were not caused by changes in levels of PC synthesizing or degrading enzymes. PE export from mitochondria to the ER during ER stress was further reduced in cells lacking Mdm10p, a component of an ER-mitochondrial tethering complex that may facilitated lipid exchange between these compartments. We also found that reducing mitochondrial PC levels induces mitophagy. In conclusion, we show that ER stress affected PE export from mitochondria to ER and the Mdm10p is important for this process. Copyright © 2016 Elsevier B.V. All rights reserved.
Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching.
Cabukusta, Birol; Köhlen, Jan A; Richter, Christian P; You, Changjiang; Holthuis, Joost C M
Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver.
Lièvremont, J P; Hill, A M; Hilly, M; Mauger, J P
Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane. Images Figure 2 Figure 3 Figure 4 Figure
Endoplasmic reticulum stress: key promoter of rosacea pathogenesis.
Melnik, Bodo C
Recent scientific interest in the pathogenesis of rosacea focuses on abnormally high facial skin levels of cathelicidin and the trypsin-like serine protease kallikrein 5 (KLK5) that cleaves the cathelicidin precursor protein into the bioactive fragment LL-37, which exerts crucial proinflammatory, angiogenic and antimicrobial activities. Furthermore, increased expression of toll-like receptor 2 (TLR2) has been identified in rosacea skin supporting the participation of the innate immune system. Notably, TLRs are expressed on sensory neurons and increase neuronal excitability linking TLR signalling to the transmission of neuroinflammatory responses. It is the intention of this viewpoint to present a unifying concept that links all known clinical trigger factors of rosacea such as UV irradiation, heat, skin irritants and special foods to one converging point: enhanced endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR). ER stress via upregulation of transcription factor ATF4 increases TLR2 expression, resulting in enhanced production of cathelicidin and KLK5 mediating downstream proinflammatory, angiogenic and antimicrobial signalling. The presented concept identifies rosacea trigger factors as environmental stressors that enhance the skin's ER stress response. Exaggerated cutaneous ER stress that stimulates the TLR2-driven inflammatory response may involve sebocytes, keratinocytes, monocyte-macrophages and sensory cutaneous neurons. Finally, all antirosacea drugs are proposed to attenuate the ER stress signalling cascade at some point. Overstimulated ER stress signalling may have evolutionarily evolved as a compensatory mechanism to balance impaired vitamin D-driven LL-37-mediated antimicrobial defenses due to lower exposure of UV-B irradiation of the northern Celtic population. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Transcriptional analysis implicates endoplasmic reticulum stress in bovine spongiform encephalopathy.
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Yue Tang
Full Text Available Bovine spongiform encephalopathy (BSE is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity.
Analysis of nelfinavir-induced endoplasmic reticulum stress.
Brüning, Ansgar
Nelfinavir (Viracept®) is an HIV protease inhibitor that has been shown to induce the endoplasmic reticulum (ER) stress reaction in human cancer cells. Although the presumed drug doses needed for an efficient ER stress reaction and ensuing apoptosis in cancer cells is somewhat higher than those prescribed for HIV-infected persons, nelfinavir represents one of the few clinically applicable ER stress-inducing agents, and is currently being tested in clinical studies on cancer patients. Therefore, this chapter describes how to obtain and use nelfinavir for in vitro and in vivo studies. In addition, methods are described that might facilitate the analysis and monitoring of the nelfinavir-induced ER stress response either in cancer cells in cell culture or in cancer tissue biopsies. These methods include various fluorescence-based ER staining techniques and the expression analysis of primary and secondary ER stress markers by immunoblotting and RT-PCR analysis. Among the several methods presented, the analysis of an unconventional XBP1 splicing, caused by the ER stress sensor IRE1, is shown to present the most sensitive and most specific marker for nelfinavir-induced ER stress. Primers and PCR conditions suitable for XBP1 PCR and splicing analysis are presented. Such a PCR-based XBP1 splicing analysis might not only be suitable to monitor nelfinavir-induced ER stress, but could also be applied in drug screening programs to test for other ER stress-inducing agents with similar activities or synergistic activities with nelfinavir. Copyright © 2011 Elsevier Inc. All rights reserved.
Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of resistance blood vessel structure and function.
Carlisle, Rachel E; Werner, Kaitlyn E; Yum, Victoria; Lu, Chao; Tat, Victor; Memon, Muzammil; No, Yejin; Ask, Kjetil; Dickhout, Jeffrey G
Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.
The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia
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Poone, Gagandip K.; Hasseldam, Henrik; Munkholm, Nina
Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response,resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an in vitro and in vivo model...... (p endoplasmic reticulum stress induced apoptosis in stroke. Ashypothermia further...... globalischemia. A stable increase in CHOP expression was observed throughout the time course (p stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours...
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Ramsgaard Thomsen, Mette
, Vivisection and Strange Metabolisms, were developed at the Centre for Information Technology and Architecture (CITA) at the Royal Danish Academy of Fine Arts in Copenhagen as a means of engaging intangible digital data with tactile physical material. As robotic membranes, they are a dual examination...
Alternative splicing of Spg7, a gene involved in hereditary spastic paraplegia, encodes a variant of paraplegin targeted to the endoplasmic reticulum.
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Giuseppe Mancuso
Full Text Available BACKGROUND: Hereditary spastic paraplegia defines a group of genetically heterogeneous diseases characterized by weakness and spasticity of the lower limbs owing to retrograde degeneration of corticospinal axons. One autosomal recessive form of the disease is caused by mutation in the SPG7 gene. Paraplegin, the product of SPG7, is a component of the m-AAA protease, a high molecular weight complex that resides in the mitochondrial inner membrane, and performs crucial quality control and biogenesis functions in mitochondria. PRINCIPAL FINDINGS: Here we show the existence in the mouse of a novel isoform of paraplegin, which we name paraplegin-2, encoded by alternative splicing of Spg7 through usage of an alternative first exon. Paraplegin-2 lacks the mitochondrial targeting sequence, and is identical to the mature mitochondrial protein. Remarkably, paraplegin-2 is targeted to the endoplasmic reticulum. We find that paraplegin-2 exposes the catalytic domains to the lumen of the endoplasmic reticulum. Moreover, endogenous paraplegin-2 accumulates in microsomal fractions prepared from mouse brain and retina. Finally, we show that the previously generated mouse model of Spg7-linked hereditary spastic paraplegia is an isoform-specific knock-out, in which mitochondrial paraplegin is specifically ablated, while expression of paraplegin-2 is retained. CONCLUSIONS/SIGNIFICANCE: These data suggest a possible additional role of AAA proteases outside mitochondria and open the question of their implication in neurodegeneration.
Triggering apoptotic death of human epidermal keratinocytes by malic Acid: involvement of endoplasmic reticulum stress- and mitochondria-dependent signaling pathways.
Hsiao, Yu-Ping; Lai, Wan-Wen; Wu, Shi-Bei; Tsai, Chung-Hung; Tang, Sheau-Chung; Chung, Jing-Gung; Yang, Jen-Hung
Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.
Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways
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Yu-Ping Hsiao
Full Text Available Malic acid (MA has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT. The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs. Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR was significantly decreased whereas extracellular acidification rate (ECAR was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.
Tomato Ribonuclease LX with the Functional Endoplasmic Reticulum Retention Motif HDEF Is Expressed during Programmed Cell Death Processes, Including Xylem Differentiation, Germination, and Senescence1
Lehmann, Karin; Hause, Bettina; Altmann, Dorit; Köck, Margret
We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX. PMID:11598219
Oxidation in HiOx-packaged pork Longissimus muscle predisposes myofibrillar and sarcoplasmic proteins to N-nitrosamine formation in nitrite-curing solution.
Yang, Hua; Meng, Peipei; Xiong, Youling L; Ma, Lizhen; Wang, Changlu; Zhu, Yingchun
The effect of meat protein in situ oxidation on the formation of N-nitrosodiethylamine (NDEA) was investigated. Fresh minced pork was untreated (Con) or treated with 700mg/kg α-tocopherol (Toc) or 300mg/kg tea polyphenols (PPE), packaged in a HiOx atmosphere (78.8% O2, 18.8% CO2, 2.4% N2), then stored at 2±1°C for up to 10days. Crude myofibrillar (MP) or sarcoplasmic (SP) protein (20mg/mL) extracted from stored meat was reacted with 43μM sodium nitrite at 80°C for 1h. Lipid oxidation was totally inhibited in PPE pork but increased in Con and Toc samples after 10days. There was significant protein oxidation (losses of sulfhydryls, formation of protein carbonyls) in both MP and SP in all samples during storage. However, the Con group suffered more extensive protein oxidation than Toc and PPE and produced more NDEA (P<0.05), indicating that protein oxidation promoted nitrosation. Copyright © 2013 Elsevier Ltd. All rights reserved.
[Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].
Wang, Cai-xia; Huang, Jian-fang; Xiang, Jun-jian; Sun, Yi-fan; Lv, Si; Guo, Jie
To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.
PARM-1 is an endoplasmic reticulum molecule involved in endoplasmic reticulum stress-induced apoptosis in rat cardiac myocytes.
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Koji Isodono
Full Text Available To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1. While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER. In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.
Curvature of double-membrane organelles generated by changes in membrane size and composition.
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Roland L Knorr
Full Text Available Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae.
Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis.
Guo, Jun-Jie; Ma, Lei-Lei; Shi, Hong-Tao; Zhu, Jian-Bing; Wu, Jian; Ding, Zhi-Wen; An, Yi; Zou, Yun-Zeng; Ge, Jun-Bo
Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis.
Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.
Wang, Yu; Wang, Yi-Li; Huang, Xia; Yang, Yang; Zhao, Ya-Jun; Wei, Cheng-Xi; Zhao, Ming
Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H2O2, ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H2O2. The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H2O2-induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.
Endoplasmic reticulum stress in pathogenesis of diabetic retinopathy and effect of calcium dobesilate
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Yu-Min Gui
Full Text Available Objective: To study the mechanism of endoplasmic reticulum stress in the pathogenesis of diabetic retinopathy and effect of calcium dobesilate. Methods: A total of 120 diabetic retinopathy patients treated in our hospital from January 2010 to September 2015 were enrolled in this article. The serum endoplasmic reticulum stress protein and interleukin protein expression levels were analyzed before and after calcium dobesilate treatment. A total of 55 cases of healthy subjects receiving physical examination in our hospital during the same period were taken as control group. Results: Serum endoplasmic reticulum stress proteins PERK, CHOP and IRE as well as interleukin proteins IL1, IL2, IL6 and IL10 expression significantly increased, serum MDA level significantly increased while SOD, CAT and GSHpx levels significantly decreased in diabetic retinopathy patients, and compared with control group (P<0.01; after calcium dobesilate treatment, above factors were significantly restored (P<0.01. Conclusions: Diabetic retinopathy is closely related to endoplasmic reticulum stress and calcium dobesilate treatment may improve diabetic retinopathy by inhibiting endoplasmic reticulum stress.
Spatial regulation of membrane fusion controlled by modification of phosphoinositides.
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Fabrice Dumas
Full Text Available Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCgamma, GTP hydrolysis and (phosphatidylinsositol-(4,5-bisphosphate (PtdIns(4,5P(2 are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.
Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes.
Andrews, J; Keegstra, K
Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum.
The Role of Endoplasmic Reticulum Stress and Unfolded Protein Response in Atherosclerosis.
Ivanova, Ekaterina A; Orekhov, Alexander N
Pathogenesis of atherosclerosis is a complex process involving several metabolic and signalling pathways. Accumulating evidence demonstrates that endoplasmic reticulum stress and associated apoptosis can be induced in the pathological conditions of atherosclerotic lesions and contribute to the disease progression. Notably, they may play a role in the development of vulnerable plaques that induce thrombosis and are therefore especially dangerous. Endoplasmic reticulum stress response is regulated by several signaling mechanisms that involve protein kinases and transcription factors. Some of these molecules can be regarded as potential therapeutic targets to improve treatment of atherosclerosis. In this review we will discuss the role of endoplasmic reticulum stress and apoptosis in atherosclerosis development in different cell types and summarize the current knowledge on potential therapeutic agents targeting molecules regulating these pathways and their possible use for anti-atherosclerotic therapy.
Endoplasmic reticulum quality control is involved in the mechanism of endoglin-mediated hereditary haemorrhagic telangiectasia.
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Bassam R Ali
Full Text Available Hereditary haemorrhagic telangiectasia (HHT is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W out of thirteen mutants in the Zona Pellucida (ZP domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional
ERLIN2 promotes breast cancer cell survival by modulating endoplasmic reticulum stress pathways
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Wang Guohui
Full Text Available Abstract Background Amplification of the 8p11-12 region has been found in approximately 15% of human breast cancer and is associated with poor prognosis. Previous genomic analysis has led us to identify the endoplasmic reticulum (ER lipid raft-associated 2 (ERLIN2 gene as one of the candidate oncogenes within the 8p11-12 amplicon in human breast cancer, particularly in the luminal subtype. ERLIN2, an ER membrane protein, has recently been identified as a novel mediator of ER-associated degradation. Yet, the biological roles of ERLIN2 and molecular mechanisms by which ERLIN2 coordinates ER pathways in breast carcinogenesis remain unclear. Methods We established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for in vitro transforming phenotypes. Next, we knocked down the ERLIN2 as well as the ER stress sensor IRE1α activity in the breast cancer cell lines to characterize the biological roles and molecular basis of the ERLIN2 in carcinogenesis. Finally, immunohistochemical staining was performed to detect ERLIN2 expression in normal and cancerous human breast tissues Results We found that amplification of the ERLIN2 gene and over expression of the ERLIN2 protein occurs in both luminal and Her2 subtypes of breast cancer. Gain- and loss-of-function approaches demonstrated that ERLIN2 is a novel oncogenic factor associated with the ER stress response pathway. The IRE1α/XBP1 axis in the ER stress pathway modulated expression of ERLIN2 protein levels in breast cancer cells. We also showed that over expression of ERLIN2 facilitated the adaptation of breast epithelial cells to ER stress by supporting cell growth and protecting the cells from ER stress-induced cell death. Conclusions ERLIN2 may confer a selective growth advantage for breast cancer cells by
Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death.
Choi, Sekyu; Quan, Xianglan; Bang, Sunhoe; Yoo, Heesuk; Kim, Jiyoung; Park, Jiwon; Park, Kyu-Sang; Chung, Jongkyeong
Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells.
Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie
Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH.
Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells.
Lee, Ah Young; Han, Young-Ah; Kim, Ji-Eun; Hong, Seong-Ho; Park, Eun-Jung; Cho, Myung-Haing
In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 μg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1α (IRE1α) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca(2+) leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress. Copyright © 2015 Elsevier Ltd. All rights reserved.
Oleosome-Associated Protein of the Oleaginous Diatom Fistulifera solaris Contains an Endoplasmic Reticulum-Targeting Signal Sequence
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Yoshiaki Maeda
Full Text Available Microalgae tend to accumulate lipids as an energy storage material in the specific organelle, oleosomes. Current studies have demonstrated that lipids derived from microalgal oleosomes are a promising source of biofuels, while the oleosome formation mechanism has not been fully elucidated. Oleosome-associated proteins have been identified from several microalgae to elucidate the fundamental mechanisms of oleosome formation, although understanding their functions is still in infancy. Recently, we discovered a diatom-oleosome-associated-protein 1 (DOAP1 from the oleaginous diatom, Fistulifera solaris JPCC DA0580. The DOAP1 sequence implied that this protein might be transported into the endoplasmic reticulum (ER due to the signal sequence. To ensure this, we fused the signal sequence to green fluorescence protein. The fusion protein distributed around the chloroplast as like a meshwork membrane structure, indicating the ER localization. This result suggests that DOAP1 could firstly localize at the ER, then move to the oleosomes. This study also demonstrated that the DOAP1 signal sequence allowed recombinant proteins to be specifically expressed in the ER of the oleaginous diatom. It would be a useful technique for engineering the lipid synthesis pathways existing in the ER, and finally controlling the biofuel quality.
Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation
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Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis, E-mail: firstname.lastname@example.com
Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.
Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers.
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Takuya Akiyama
Full Text Available Endoplasmic reticulum (ER stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon.
Oxidative stress induces monocyte necrosis with enrichment of cell-bound albumin and overexpression of endoplasmic reticulum and mitochondrial chaperones.
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Haiping Tang
Full Text Available In the present study, monocytes were treated with 5-azacytidine (azacytidine, gossypol or hydrogen peroxide to induce cell death through oxidative stress. A shift from apoptotic to necrotic cell death occurred when monocytes were treated with 100 µM azacytidine for more than 12 hours. Necrotic monocytes exhibited characteristics, including enrichment of cell-bound albumin and up-regulation of endoplasmic reticulum (ER- and mitochondrial-specific chaperones to protect mitochondrial integrity, which were not observed in other necrotic cells, including HUH-7, A2780, A549 and HOC1a. Our results show that the cell-bound albumin originates in the culture medium rather than from monocyte-derived hepatocytes, and that HSP60 is a potential binding partner of the cell-bound albumin. Proteomic analysis shows that HSP60 and protein disulfide isomerase are the most abundant up-regulated mitochondrial and ER-chaperones, and that both HSP60 and calreticulin are ubiquitinated in necrotic monocytes. In contrast, expression levels of the cytosolic chaperones HSP90 and HSP71 were down-regulated in the azacytidine-treated monocytes, concomitant with an increase in the levels of these chaperones in the cell culture medium. Collectively, our results demonstrates that chaperones from different organelles behave differently in necrotic monocytes, ER- and mitochondrial chaperones being retained and cytosolic and nuclear chaperones being released into the cell culture medium through the ruptured cell membrane. HSP60 may serve as a new target for development of myeloid leukemia treatment.
Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers.
Akiyama, Takuya; Oishi, Kenji; Wullaert, Andy
Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon.
Apoptosis and necrosis induced by novel realgar quantum dots in human endometrial cancer cells via endoplasmic reticulum stress signaling pathway.
Wang, Huan; Liu, Zhengyun; Gou, Ying; Qin, Yu; Xu, Yaze; Liu, Jie; Wu, Jin-Zhu
Realgar (AS4S4) has been used in traditional medicines for malignancy, but the poor water solubility is still a major hindrance to its clinical use. Realgar quantum dots (RQDs) were therefore synthesized with improved water solubility and bioavailability. Human endometrial cancer JEC cells were exposed to various concentrations of RQDs to evaluate their anticancer effects and to explore mechanisms by the MTT assay, transmission electron microscopy (TEM), flow cytometry, real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Results revealed that the highest photoluminescence quantum yield of the prepared RQDs was up to approximately 70%, with the average size of 5.48 nm. RQDs induced antipro-liferative activity against JEC cells in a concentration-dependent manner. In light microscopy and TEM examinations, RQDs induced vacuolization and endoplasmic reticulum (ER) dilation in JEC cells in a concentration-dependent manner. ER stress by RQDs were further confirmed by increased expression of GADD153 and GRP78 at both mRNA and protein levels. ER stress further led to JEC cell apoptosis and necrosis, as evidenced by flow cytometry and mitochondrial membrane potential detection. Our findings demonstrated that the newly synthesized RQDs were effective against human endometrial cancer cells. The underlying mechanism appears to be, at least partly, due to ER stress leading to apoptotic cell death and necrosis.
Metabolomics and lipidomics profiling of a combined mitochondrial plus endoplasmic reticulum fraction of human fibroblasts: a robust tool for clinical studies.
Veyrat-Durebex, Charlotte; Bocca, Cinzia; Chupin, Stéphanie; Kouassi Nzoughet, Judith; Simard, Gilles; Lenaers, Guy; Reynier, Pascal; Blasco, Hélène
Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles functions, dysfunctions and interactions. Here, we developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with an excellent reproducibility. Seventy two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12 % among which many specific of the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.
Penfluridol induces endoplasmic reticulum stress leading to autophagy in pancreatic cancer.
Ranjan, Alok; German, Nadezhda; Mikelis, Constantinos; Srivenugopal, Kalkunte; Srivastava, Sanjay K
Pancreatic cancer is one of the most aggressive and difficult to treat cancers. Experimental and clinical evidence suggests that high basal state autophagy in pancreatic tumors could induce resistance to chemotherapy. Recently, we have demonstrated that penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis both in vitro and in vivo; however, the mechanism of autophagy induction by penfluridol was not clear. Several studies have established that endoplasmic reticulum stress could lead to autophagy and inhibit tumor progression. In this study, we demonstrated that penfluridol induced endoplasmic reticulum stress in BxPC-3, AsPC-1, and Panc-1 pancreatic cancer cell lines as indicated by upregulation of endoplasmic reticulum stress markers such as binding protein (BIP), C/EBP homologous protein (CHOP) and inositol requiring 1α (IRE1α) after treatment with penfluridol in a concentration-dependent manner. Inhibiting endoplasmic reticulum stress by pretreatment with pharmacological inhibitors such as sodium phenylbutyrate and mithramycin or by silencing CHOP using CHOP small interfering RNA, blocked penfluridol-induced autophagy. These results clearly indicate that penfluridol-induced endoplasmic reticulum stress lead to autophagy in our model. Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP, and IRE1α expression in the tumor lysates from penfluridol-treated mice as compared to tumors from control mice. Altogether, our study establishes that penfluridol-induced endoplasmic reticulum stress leads to autophagy resulting in reduced pancreatic tumor growth. Our study opens a new therapeutic target for advanced chemotherapies against pancreatic cancer.
The Arabidopsis thaliana AAA protein CDC48A interacts in vivo with the somatic embryogenesis receptor-like kinase 1 receptor at the plasma membrane.
Aker, J.C.M.; Borst, J.W.; Karlova, R.B.; Vries, de S.C.
Fluorescent cell division cycle (CDC)48 proteins were studied in living plant protoplasts. CDC48A and somatic embryogenesis receptor like kinase 1 (SERK1) were found to co-localize in the endoplasmatic reticulum (ER) and at the plasma membrane (PM), but not in endosomal compartments. Fluorescent
PDMP blocks brefeldin A-induced retrograde membrane transport from Golgi to ER : Evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism
Kok, JW; Babia, T; Filipeanu, CM; Nelemans, A; Egea, G; Hoekstra, D
In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP
Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells
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Chiao-I. Lai
Full Text Available Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy.
Mitochondrial pathway and endoplasmic reticulum stress participate in the photosensitizing effectiveness of AE-PDT in MG63 cells.
Li, Kai-Ting; Chen, Qing; Wang, Da-Wu; Duan, Qin-Qin; Tian, Si; He, Juan-Wen; Ou, Yun-Sheng; Bai, Ding-Qun
Photodynamic therapy (PDT) is a promising treatment in cancer therapy, with a photosensitizer activated by visible light. Aloe-emodin (AE) is a promising photosensitive agent. In this study, the photosensitizing effects and possible mechanisms of AE-PDT in MG63 cells were evaluated. The efficiency of AE-PDT was analyzed by MTT assay. The mode of cell death was investigated by Hoechst 33,342 staining and flow cytometer. The intracellular distribution of AE was detected with confocal microscopy. The formation of reactive oxygen species (ROS) was detected by DCFH-DA. The mitochondrial membrane potential (MMP) was measured by Rhodamine 123. The expression of proteins including cytochrome c, caspase-3, -9, and -12, CHOP and GRP78 was detected by western blot. Apoptosis is the primary mode of cell death in our study, which occurs in a manner of depending on AE concentration and irradiation dose. Confocal microscopy showed that AE was primarily localized on the mitochondria and endoplasmic reticulum (ER) of MG63 cells. AE-PDT resulted in rapid increases of intracellular ROS production, which reached a peak at 2 h, followed by declining of mitochondrial membrane potential, releasing of cytochrome c from mitochondria into the cytoplasm, and up-regulation of caspase-3, -9, and -12, CHOP and GRP78. These results suggest that death of MG63 cells induced by AE-PDT is triggered by ROS. Meanwhile, Mitochondria and ER serve as the subcellular targets, which are responsible for AE-PDT-induced death of MG63 cells. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
Realgar quantum dots induce apoptosis and necrosis in HepG2 cells through endoplasmic reticulum stress.
Qin, Y U; Wang, Huan; Liu, Zheng-Yun; Liu, Jie; Wu, Jin-Zhu
Realgar (As 4 S 4 ) has been used in traditional Chinese medicines for treatment of malignancies. However, the poor water solubility of realgar limits its clinical application. To overcome this problem, realgar quantum dots (RQDs; 5.48±1.09 nm) were prepared by a photoluminescence method. The mean particle size was characterized by high-resolution transmission electron microscopy and scanning electron microscopy. Our recent studies revealed that the RQDs were effective against tumor growth in tumor-bearing mice without producing apparent toxicity. The present study investigated their anticancer effects and mechanisms in human hepatocellular carcinoma (HepG2) cells. The HepG2 cells and human normal liver (L02) cells were used to determine the cytotoxicity of RQDs. The portion of apoptotic and dead cells were measured by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Apoptosis-related proteins and genes were examined by western blot analysis and reverse transcription-quantitative polymerase chain reaction, and the mitochondrial membrane potential was assayed by confocal microscope with JC-1 as a probe. RQDs exhibited cytotoxicity in a concentration-dependent manner and HepG2 cells were more sensitive compared with normal L02 cells. At 15 µg/ml, 20% of the cells were apoptotic, while 60% of the cells were necrotic at 30 µg/ml. The anti-apoptosis protein Bcl-2 was dose-dependently decreased, while pro-apoptotic protein Bax was increased. There was a loss of mitochondrial membrane potential and expression of the stress genes C/EBP-homologous protein 10 and glucose-regulated protein 78 was increased by RQDs. RQDs were effective in the inhibition of HepG2 cell proliferation and this effect was due to induction of apoptosis and necrosis through endoplasmic reticulum stress.
[Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy].
Yu, Y; Zhou, C L; Yu, T T; Han, X J; Shi, H Y; Wang, H Z; Shen, J J; He, J
Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, Preticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.04, 1.17±0.15, 1.33±0.13, 1.73±0
Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress.
Yang, Jian-Wen; Hu, Zhi-Ping
Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.
Endoplasmic reticulum proteins quality control and the unfolded protein response: the regulative mechanism of organisms against stress injuries.
Fu, Xi Ling; Gao, Dong Sheng
The endoplasmic reticulum is the cellular compartment in which secretory proteins are synthesized and folded. Perturbations of endoplasmic reticulum homeostasis lead to the accumulation of unfolded proteins. The activation of the unfolded protein response during endoplasmic reticulum stress transmits information about the status of protein folding to the cytosol and nucleus. The unfolded protein response leads to the upregulation of genes encoding endoplasmic reticulum chaperones, attenuation of translation, and initiation of the endoplasmic reticulum quality control system to restore endoplasmic reticulum homeostasis. When the unfolded protein response is insufficient to rebuild the steady state in endoplasmic reticulum, the programmed cell death or apoptosis would be initiated, by triggering cell injuries, even to cell death through apoptosis signals. In this review, we briefly outline research on the chaperones and foldases conserved in eukaryotes and plants, and describe the general principles and mechanisms of the endoplasmic reticulum quality control and the unfolded protein response. We describe the current models for the molecular mechanism of the unfolded protein response in plants, and emphasize the role of inositol requiring enzyme-1-dependent network in the unfolded protein response. Finally, we give a general overview of the directions for future research on the unfolded protein response in plants and its role in the response to environmental stresses. © 2014 International Union of Biochemistry and Molecular Biology.
Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor
Fueller, Julia; Egorov, Mikhail V.; Walther, Kirstin A.; Sabet, Ola; Mallah, Jana; Grabenbauer, Markus; Kinkhabwala, Ali
The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B's mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B's insertion into the ER membrane through heterologous expression of PTP1B's tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states. PMID:26431424
An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids
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Harriet Allison
Full Text Available African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate 'invariant glycoproteins' (IGPs. IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism.
An allosteric model of the molecular interactions of excitation- contraction coupling in skeletal muscle
A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88- 118), and analogous to one proposed by Marks and Jones for voltage- dependent Ca ch...
Calmodulin kinase is a molecular switch for cardiac excitation –contraction coupling
Wu, Yuejin; Colbran, Roger J.; Anderson, Mark E.
Signaling between cell membrane-bound L-type Ca2+ channels (LTCC) and ryanodine receptor Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation–contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for a...
Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex
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Coudroy, Gwénaëlle; Gburek, Jakub; Kozyraki, Renata
fragments, including a functional "mini" version of cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of cubilin, did not appear at the plasma membrane but instead were......-localized with cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both...... units contribute to the processing of the cubilin-AMN complex to the apical membrane: AMN interacts with the EGF domains of cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular cubilin molecule is responsible for apical...
Membrane trafficking pathways and their roles in plant-microbe interactions.
Inada, Noriko; Ueda, Takashi
Membrane trafficking functions in the delivery of proteins that are newly synthesized in the endoplasmic reticulum (ER) to their final destinations, such as the plasma membrane (PM) and the vacuole, and in the internalization of extracellular components or PM-associated proteins for recycling or degradative regulation. These trafficking pathways play pivotal roles in the rapid responses to environmental stimuli such as challenges by microorganisms. In this review, we provide an overview of the current knowledge of plant membrane trafficking and its roles in plant-microbe interactions. Although there is little information regarding the mechanism of pathogenic modulation of plant membrane trafficking thus far, recent research has identified many membrane trafficking factors as possible targets of microbial modulation.
Mitochondria-Associated Membranes and ER Stress.
van Vliet, Alexander R; Agostinis, Patrizia
The endoplasmic reticulum (ER) is a crucial organelle for coordinating cellular Ca(2+) signaling and protein synthesis and folding. Moreover, the dynamic and complex membranous structures constituting the ER allow the formation of contact sites with other organelles and structures, including among others the mitochondria and the plasma membrane (PM). The contact sites that the ER form with mitochondria is a hot topic in research, and the nature of the so-called mitochondria-associated membranes (MAMs) is continuously evolving. The MAMs consist of a proteinaceous tether that physically connects the ER with mitochondria. The MAMs harness the main functions of both organelles to form a specialized subcompartment at the interface of the ER and mitochondria. Under homeostatic conditions, MAMs are crucial for the efficient transfer of Ca(2+) from the ER to mitochondria, and for proper mitochondria bioenergetics and lipid synthesis. MAMs are also believed to be the master regulators of mitochondrial shape and motility, and to form a crucial site for autophagosome assembly. Not surprisingly, MAMs have been shown to be a hot spot for the transfer of stress signals from the ER to mitochondria, most notably under the conditions of loss of ER proteostasis, by engaging the unfolded protein response (UPR). In this chapter after an introduction on ER biology and ER stress, we will review the emerging and key signaling roles of the MAMs, which have a root in cellular processes and signaling cascades coordinated by the ER.
4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.
Liu, Liangming; Wu, Huiling; Zang, JiaTao; Yang, Guangming; Zhu, Yu; Wu, Yue; Chen, Xiangyun; Lan, Dan; Li, Tao
Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. Laboratory investigation. State Key Laboratory of Trauma, Burns and Combined Injury. Sprague-Dawley rats. Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. Anti
KDEL Receptors Assist Dengue Virus Exit from the Endoplasmic Reticulum
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Ming Yuan Li
Full Text Available Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV interacts with KDEL receptors (KDELR, which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs. Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking.
Effects of ginger extract on smooth muscle activity of sheep reticulum and rumen
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Amin Mamaghani
Full Text Available Reticulorumen hypomotility leads to the impaired physiologic functions of the digestive tract. Prokinetic action of ginger has been demonstrated in the laboratory animals and human. The aim of this study was to evaluate the effect of hydroalcoholic extract of ginger on contraction and motility of reticulum and rumen of ruminants. Collected samples of reticulum and rumen from eight sheep were investigated in vitro. The extract at the concentration of 0.1 and 1.0 mg L-1 had no effect on any preparations. Contraction of reticulum and rumen preparations was occurred at 10.0 and 100 mg L-1 concentrations (p < 0.05. Concentration of 1000 mg L-1 caused a relaxation in preparations contracted with 10.0 and 100 mg L-1. Likewise, the concentration of 1000 mg L-1 significantly (p < 0.05 inhibited ACh-induced contraction in both tissues. Six sheep were involved in electromyographic study. Administration of 40 mg kg-1 of the extract increased the overall frequency of contractions of the reticulum and rumen at the subsequent three days with the prominent increase at the second day (p < 0.05. Results of in vitro study indicated that hydroalcoholic extract of ginger contained spasmogenic and spasmolytic constituents. The results in vivo study represented evidences that the extract may have stimulant effect on reticulorumen motility in 40 mg kg-1 concentration.
Assembly of rat hepatic very low density lipoproteins in the endoplasmic reticulum
Rusiñol, A; Verkade, H; Vance, J E
The intracellular site of assembly of hepatic very low density lipoproteins has been investigated. Two endoplasmic reticulum fractions and Golgi vesicles (relatively free from endosomal contamination) were isolated from rat liver and the luminal contents were released. The apoB-containing entities
Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis
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Alexandria Hughes
Full Text Available Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.
Lipid-induced endoplasmic reticulum stress in X-linked adrenoleukodystrophy.
van de Beek, Malu-Clair; Ofman, Rob; Dijkstra, Inge; Wijburg, Frits; Engelen, Marc; Wanders, Ronald; Kemp, Stephan
X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease that is caused by mutations in the ABCD1 gene and characterized by elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues, with the most pronounced increase in the central nervous system. Virtually all male patients develop adrenal insufficiency and myelopathy (adrenomyeloneuropathy), but a subset develops a fatal cerebral demyelinating disease (known as cerebral ALD). Female patients may also develop myelopathy, but adrenal insufficiency or leukodystrophy are very rare. ALD has been associated with mitochondrial dysfunction, oxidative stress and bioenergetic failure, but the mechanism by which VLCFA accumulation triggers these effects has not been resolved thus far. In this study, we used primary human fibroblasts from normal subjects and ALD patients to investigate whether VLCFA can induce endoplasmic reticulum stress. We show that saturated VLCFA (C26:0) induce endoplasmic reticulum stress in fibroblasts from ALD patients, but not in controls. Furthermore, there is a clear correlation between the chain-length of the fatty acid and the induction of endoplasmic reticulum stress. Exposure of ALD fibroblasts to C26:0, resulted in increased expression of additional endoplasmic reticulum stress markers (EDEM1, GADD34 and CHOP) and in lipoapoptosis. This new insight into the underlying mechanism of VLCFA-induced toxicity is of great importance for the development of a disease modifying treatment for ALD aimed at the normalization of VLCFA levels in tissues. Copyright © 2017 Elsevier B.V. All rights reserved.
Role of Endoplasmic Reticulum Stress in Silica-induced Apoptosis in RAW264.7 Cells.
Hu, Yong Bin; Wu, Xia; Qin, Xiao Feng; Wang, Lei; Pan, Pin Hua
We investigated the role of endoplasmic reticulum stress (ERS) in silica-induced apoptosis in alveolar macrophages in vitro. RAW264.7 cells were incubated with 200 μg/mL silica for different time periods. Cell viability was assayed by the MTT assay. Cell apoptosis was evaluated by DAPI staining, flow cytometry analysis, and Western blot analysis of caspase-3. Morphological changes in the endoplasmic reticulum were observed by transmission electron microscopy. The expression of ERS markers binding protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) was examined by Western blotting and real-time PCR. As an inhibitor of ERS, 4-phenylbutyric acid (4-PBA) was used in the experiments. Silica exposure induced nuclear condensation and caspase-3 expression in RAW264.7 cells. The number of apoptotic cells increased after silica exposure in a time-dependent manner. Silica treatment induced expansion of the endoplasmic reticulum. In addition, the expression of BiP and CHOP increased in silica-stimulated cells. Furthermore, 4-PBA treatment inhibited silica-induced endoplasmic reticulum expansion and the expression of BiP and CHOP. Moreover, 4-PBA treatment attenuated nuclear condensation, reduced apoptotic cells, and downregulated caspase-3 expression in silica-stimulated cells. Silica-induced ERS is involved in the apoptosis of alveolar macrophages. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.
Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.
Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom
Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.
Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated
Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D
Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by
Comparing Galactic Center MSSM dark matter solutions to the Reticulum II gamma-ray data
Achterberg, A.; van Beekveld, M.; Beenakker, W.; Caron, S.; Hendriks, L.
Observations with the Fermi Large Area Telescope (LAT) indicate a possible small photon signal originating from the dwarf galaxy Reticulum II that exceeds the expected background between 2 GeV and 10 GeV . We have investigated two specific scenarios for annihilating WIMP dark matter within the
Grab a Golgi: Laser trapping of golgi bodies reveals in vivo Interactions with the endoplasmic reticulum
Sparkes, I.A.; Ketelaar, T.; Ruijter, de N.C.A.; Hawes, C.
In many vacuolate plant cells individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi
Endoplasmic reticulum stress suppressive compounds from the edible mushroom Mycoleptodonoides aitchisonii.
Choi, Jae-Hoon; Suzuki, Tomohiro; Okumura, Hiroshi; Noguchi, Keiichi; Kondo, Mitsuru; Nagai, Kaoru; Hirai, Hirofumi; Kawagishi, Hirokazu
Two novel compounds, 1 and 7, along with six known compounds (2-6 and 8), were isolated from the edible mushroom Mycoleptodonoides aitchisonii (bunaharitake in Japanese). The structures of the new compounds were determined by the interpretation of spectroscopic data. Compounds 1-4 and 6-8 showed protective activity against endoplasmic reticulum stress-dependent cell death.
Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response
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Hansen, Henning Gram; Schmidt, Jonas Damgard; Soltoft, Cecilie Lutzen
Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the o...
Increased endoplasmic reticulum stress in mouse osteocytes with aging alters Cox-2 response to mechanical stimuli
Chalil, S.; Jaspers, R.T.; Manders, R.J.; Klein-Nulend, J.; Bakker, A.D.; Deldicque, L.
Aging reduces bone mass as well as the anabolic response of bone to mechanical stimuli, resulting in osteopenia. Endoplasmic reticulum (ER) stress impairs the response of myogenic cells to anabolic stimuli, and is involved in sarcopenia, but whether ER stress also contributes to osteopenia is
Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast
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Hou, Jin; Tang, Hongting; Liu, Zihe
In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects...
The plasma membrane as a capacitor for energy and metabolism
Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini
When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520
Facilitative plasma membrane transporters function during ER transit.
Takanaga, Hitomi; Frommer, Wolf B
Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na(+)-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.
Hoppe, Jens
The classical dynamics of M-dimensional extended objects arising from stationary points of the world volume swept out in space time is discussed from various points of view. A introduction to the Hamiltonian mechanics of bosonic compact M(em)branes is given, emphasing the diversity of the different formulations and gauge choices. For moving hypersurfaces, a graph description—including its nonlinear realization of Lorentz invariance—and hydrodynamic formulations (in light-cone coordinates as well as when choosing the time coordinate of a Lorentz observer as the dependent variable) are presented. A matrix regularization for M = 2 (existing for all topologies) is explained in detail for the 2-sphere, as well as multilinear formulations for M > 2. The recently found dynamical symmetry that exists for all M and related reconstruction algebras are covered, just as some explicit solutions of the level-set equations.
Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells
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Cardozo, Alessandra K; Ortis, Fernanda; Storling, Joachim
Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by microar......Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown......, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding......, beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol...
Dancing with the Stars: Using Image Analysis to Study the Choreography of the Endoplasmic Reticulum and Its Partners and of Movement Within Its Tubules.
Griffing, Lawrence R
In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or nonmoving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topology, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to nonmoving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that are moving rapidly and streaming through the cell, the image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.
Zhang, Jintao; Yi, Man; Zha, Longying; Chen, Siqiang; Li, Zhijia; Li, Cheng; Gong, Mingxing; Deng, Hong; Chu, Xinwei; Chen, Jiehua; Zhang, Zheqing; Mao, Limei; Sun, Suxia
Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (si
Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.
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Jintao Zhang
Full Text Available Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.Human colorectal cancer cell lines (HCT-116 and HT-29 were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining, and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II, beclin-1, and autophagocytosis-associated protein (Atg3. The autophagy inhibitors 3-methyladenine (3-MA and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin and genetic
Anaerobic membrane bioreactors: Are membranes really necessary?
Davila, M.; Kassab, G.; Klapwijk, A.; Lier, van J.B.
Membranes themselves represent a significant cost for the full scale application of anaerobic membrane bioreactors (AnMBR). The possibility of operating an AnMBR with a self-forming dynamic membrane generated by the substances present in the reactor liquor would translate into an important saving. A
αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form
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Le Parc Annabelle
Full Text Available Abstract Background Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes. Conclusions These experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.
Sensitivity of Rabbit Ventricular Action Potential and Ca2+ Dynamics to Small Variations in Membrane Currents and Ion Diffusion Coefficients
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Yuan Hung Lo
Full Text Available Little is known about how small variations in ionic currents and Ca2+ and Na+ diffusion coefficients impact action potential and Ca2+ dynamics in rabbit ventricular myocytes. We applied sensitivity analysis to quantify the sensitivity of Shannon et al. model (Biophys. J., 2004 to 5%–10% changes in currents conductance, channels distribution, and ion diffusion in rabbit ventricular cells. We found that action potential duration and Ca2+ peaks are highly sensitive to 10% increase in L-type Ca2+ current; moderately influenced by 10% increase in Na+-Ca2+ exchanger, Na+-K+ pump, rapid delayed and slow transient outward K+ currents, and Cl− background current; insensitive to 10% increases in all other ionic currents and sarcoplasmic reticulum Ca2+ fluxes. Cell electrical activity is strongly affected by 5% shift of L-type Ca2+ channels and Na+-Ca2+ exchanger in between junctional and submembrane spaces while Ca2+-activated Cl−-channel redistribution has the modest effect. Small changes in submembrane and cytosolic diffusion coefficients for Ca2+, but not in Na+ transfer, may alter notably myocyte contraction. Our studies highlight the need for more precise measurements and further extending and testing of the Shannon et al. model. Our results demonstrate usefulness of sensitivity analysis to identify specific knowledge gaps and controversies related to ventricular cell electrophysiology and Ca2+ signaling.
Role of prostate apoptosis response 4 in translocation of GRP78 from the endoplasmic reticulum to the cell surface of trophoblastic cells.
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Marie Cohen
Full Text Available Glucose-regulated protein 78 (GRP78 is an endoplasmic reticulum (ER molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4, a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.
Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells[W][OA
Leitz, Guenther; Kang, Byung-Ho; Schoenwaelder, Monica E.A.; Staehelin, L. Andrew
The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells. PMID:19276442
Piperine, an alkaloid from black pepper, inhibits growth of human colon cancer cells via G1 arrest and apoptosis triggered by endoplasmic reticulum stress.
Yaffe, Paul B; Power Coombs, Melanie R; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W
Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75-150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT-29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin-dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up-regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine-treated HT-29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP-ribose) polymerase-1, as well as caspase activation and reduced apoptosis in the presence of the pan-caspase inhibitor zVAD-FMK. Increased expression of the endoplasmic reticulum stress-associated proteins inositol-requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine-treated HT-29 cells. Furthermore, piperine inhibited colony formation by HT-29 cells, as well as the growth of HT-29 spheroids. Cell cycle arrest and endoplasmic reticulum stress-associated apoptosis following piperine treatment of HT-29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer. © 2014 Wiley Periodicals, Inc.
The endoplasmic reticulum stress-inducible protein, Herp, is a potential triggering antigen for anti-DNA response.
Hirabayashi, Yasuhiko; Oka, Yumiko; Ikeda, Tomoko; Fujii, Hiroshi; Ishii, Tomonori; Sasaki, Takeshi; Harigae, Hideo
Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.
APO866 Increases Antitumor Activity of Cyclosporin-A by Inducing Mitochondrial and Endoplasmic Reticulum Stress in Leukemia Cells.
Cagnetta, Antonia; Caffa, Irene; Acharya, Chirag; Soncini, Debora; Acharya, Prakrati; Adamia, Sophia; Pierri, Ivana; Bergamaschi, Micaela; Garuti, Anna; Fraternali, Giulio; Mastracci, Luca; Provenzani, Alessandro; Zucal, Chiara; Damonte, Gianluca; Salis, Annalisa; Montecucco, Fabrizio; Patrone, Franco; Ballestrero, Alberto; Bruzzone, Santina; Gobbi, Marco; Nencioni, Alessio; Cea, Michele
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered. ©2015 American Association for Cancer Research.
Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.
Osterrieder, Anne; Sparkes, Imogen A; Botchway, Stan W; Ward, Andy; Ketelaar, Tijs; de Ruijter, Norbert; Hawes, Chris
The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants
Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)
To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.
Redox-sensitive endoplasmic reticulum stress and autophagy at rostral ventrolateral medulla contribute to hypertension in spontaneously hypertensive rats.
Chao, Yung-Mei; Lai, Ming-Derg; Chan, Julie Y H
Perturbations of proper functions of the endoplasmic reticulum (ER) cause accumulation of misfolded or unfolded proteins in the cell, creating a condition known as ER stress. Prolonged ER stress has been implicated in hypertension. Oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside, plays a pivotal role in neurogenic hypertension. This study aimed to evaluate the contribution of ER stress in RVLM to oxidative stress-associated hypertension and delineate the underlying molecular mechanisms. The expression of glucose-regulated protein 78 kDa and the phosphorylation of protein kinase RNA-like ER kinase-translation initiation factor α, 2 major protein markers of ER stress, were augmented in RVLM and preceded the development of hypertensive phenotype in spontaneously hypertensive rats. In RVLM of spontaneously hypertensive rats, stabilizing ER stress by salubrinal promoted antihypertension, and scavenging the reactive oxygen species by tempol reduced the augmented ER stress. Furthermore, induction of oxidative stress by angiotensin II induced ER stress in RVLM, and induction of ER stress by tunicamycin in RVLM induced pressor response in normotensive Wistar-Kyoto rats. Autophagy, as reflected by the expression of lysosome-associated membrane protein-2 and microtubule-associated protein 1 light chain 3-II (LC3-II), was significantly increased in RVLM of spontaneously hypertensive rats and was abrogated by salubrinal. In addition, inhibition of autophagy or silencing LC3-II gene in RVLM resulted in antihypertension in spontaneously hypertensive rats. These results suggest that redox-sensitive induction of ER stress and activation of autophagy in RVLM contribute to oxidative stress-associated neurogenic hypertension.
Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation*
Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel
Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD. PMID:24711457
Dissociation of Effects on Protein Synthesis and Ribosomes from Membrane Changes Induced by Carbon Tetrachloride
Farber, Emmanuel; Liang, Hilda; Shinozuka, Hisashi
Pretreatment of rats with cycloheximide protects the liver against ribosome changes but not against alterations in the endoplasmic reticulum membrane induced by the administration of CCl4, as observed both biochemically and ultrastructurally. Another inhibitor of protein synthesis, tenuazonic acid, has effects similar to cycloheximide. Neither compound has any apparent effect when given after the administration of CCl4 at a time at which a major shift in ribosome distribution from polysomes to ribosome monomers or subunits has already occurred. Thus, it appears that the ribosomes are not susceptible to damage by CCl4, or more probably an active metabolic derivative of CCl4, when they are organized as polysomes but only when they are present as monomers or subunits. Also, unlike the situation with ethionine or puromycin, the damage to the protein synthetic system of the ribosomes induced in the liver by CCl4 appears to be irreversible. In contrast to cycloheximide or tenuazonic acid, N,N'-diphenyl-p-phenylenediamine (DPPD) protects the liver against CCl4-induced changes in both ribosomes and endoplasmic reticulum. These observations suggest that the changes in the ribosome-protein synthetic system induced in the liver by treatment with CCl4 are not the direct result of damage to the membranes of the endoplasmic reticulum but are either an indirect effect of such damage or are unrelated to it. These possibilities are discussed in terms of three proposed working hypotheses. ImagesFig 5Fig 6Fig 1Fig 2Fig 7Fig 3Fig 4 PMID:5133520
Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans
Douglas, Lois M.; Konopka, James. B.
Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878
Hypotonic stress-induced calcium signaling in Saccharomyces cerevisiae involves TRP-like transporters on the endoplasmic reticulum membrane.
Rigamonti, M; Groppi, S; Belotti, F; Ambrosini, R; Filippi, G; Martegani, E; Tisi, R
Saccharomyces cerevisiae cells respond to hypotonic stress (HTS) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. Calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and Cch1-, but not Mid1-, driven manner. HTS-induced calcium release is also negatively regulated by the ER protein Cls2 and involves a poorly characterized protein, FLC2/YAL053W gene product, previously proposed to be required for FAD transport in the ER, albeit, due to its molecular features, it was also previously classified as an ion transporter. A computational analysis revealed that this gene and its three homologs in S. cerevisiae, together with previously identified Schizosaccharomyces pombe pkd2 and Neurospora crassa calcium-related spray protein, belong to a fungal branch of TRP-like ion transporters related to human mucolipin and polycystin 2 calcium transporters. Moreover, disruption of FLC2 gene confers severe sensitivity to Calcofluor white and hyper-activation of the cell wall integrity MAPK cascade, suggesting a role in cell wall maintenance as previously suggested for the fission yeast homolog. Perturbation in cytosolic resting calcium concentration and hyper-activation of calcineurin in exponentially growing cells suggest a role for this transporter in calcium homeostasis in yeast. Copyright © 2014 Elsevier Ltd. All rights reserved.
25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes
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Olsen, Brett N.; Schlesinger, Paul H.; Ory, Daniel S.; Baker, Nathan A.
Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work, using molecular dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers to examine interactions between 25-HC and cholesterol in the same bilayer. When added to cholesterol-containing membranes, 25-HC causes larger changes in membrane structure than when added to cholesterol-free membranes, demonstrating interactions between the two sterols. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and therefore its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These interactions provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through direct modulation of cholesterol availability.
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Rubin, Philip; Levitt, Seymour H.
Case histories of two patients treated with colloidal radiogold for diffuse reticulum cell sarcoma are presented. Further analysis of the method is suggested by the unusually long survival time of one of the patients. It was concluded that, although external radiotherapy remains the treatment of choice in localized reticulum cell sarcoma, intravenous colloidal radiogold may be a useful agent in lymphosarcomas with diffuse minute neoplastic liver and spleen involvements. Intravenous colloidal radiogold can produce bone marrow depression and thrombocytopenia which can lead to death. This factor tends to argue against therapeutic use of the agent. It is suggested that no more than 50 mC Au/sup 198/ intravenously should be used for treatment of this disease. (R.M.G.)
An Involvement of Oxidative Stress in Endoplasmic Reticulum Stress and Its Associated Diseases
Bhandary, Bidur; Marahatta, Anu; Kim, Hyung-Ryong; Chae, Han-Jung
The endoplasmic reticulum (ER) is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS). Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress. PMID:23263672
CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.
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Manuela Caputo
Full Text Available The DNA repair protein Cockayne syndrome group B (CSB has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.
CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.
Caputo, Manuela; Balzerano, Alessio; Arisi, Ivan; D'Onofrio, Mara; Brandi, Rossella; Bongiorni, Silvia; Brancorsini, Stefano; Frontini, Mattia; Proietti-De-Santis, Luca
The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.
Protein accumulation in the endoplasmic reticulum as a non-equilibrium phase transition.
Budrikis, Zoe; Costantini, Giulio; La Porta, Caterina A M; Zapperi, Stefano
Several neurological disorders are associated with the aggregation of aberrant proteins, often localized in intracellular organelles such as the endoplasmic reticulum. Here we study protein aggregation kinetics by mean-field reactions and three dimensional Monte carlo simulations of diffusion-limited aggregation of linear polymers in a confined space, representing the endoplasmic reticulum. By tuning the rates of protein production and degradation, we show that the system undergoes a non-equilibrium phase transition from a physiological phase with little or no polymer accumulation to a pathological phase characterized by persistent polymerization. A combination of external factors accumulating during the lifetime of a patient can thus slightly modify the phase transition control parameters, tipping the balance from a long symptomless lag phase to an accelerated pathological development. The model can be successfully used to interpret experimental data on amyloid-β clearance from the central nervous system.
Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity
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Benaim, Gustavo, E-mail: firstname.lastname@example.com [Instituto de Estudios Avanzados (IDEA), Caracas (Venezuela, Bolivarian Republic of); Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Pimentel, Adriana A., E-mail: email@example.com [Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Felibertt, Pimali [Facultad de Ciencias, Universidad de Carabobo, Valencia (Venezuela, Bolivarian Republic of); Mayora, Adriana [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Colman, Laura [Instituto Pasteur de Montevideo, Montevideo (Uruguay); Sojo, Felipe [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Rojas, Héctor [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas (Venezuela, Bolivarian Republic of); De Sanctis, Juan B. [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of)
The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.
[Effect of fluoride on the expression of endoplasmic reticulum chaperone in ameloblast of rat incisor].
Zhang, Kai-qiang; Zhang, Ying; Liu, Lu; Gu, He-feng; Ma, Lin
To investigate the effect of different concentrations of fluoride on the expression of endoplasmic reticulum chaperone, and to explore the mechanism of dental fluorosis in rat. Thirty Wistar rats were randomly divided into 3 groups. Immunohistochemistry was used to detect the expression of CRT, GRP78, XBP-1 and caspase-12 in rat incisors. Metamorph microscope images analysis system and SPSS 13.0 software package was used to analyze the data. Typical features of dental fluorosis were found in the fluoride group. Results of immunohistochemistry showed that CRT (F=238.6, Pameloblasts and in status of endoplasmic reticulum stress and caspase-12 plays an important role during ameloblast apoptosis. Supported by National Natural Science Foundation of China (81072245) and Natural Science Foundation of Liaoning Province (20102278).
A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells
Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying
As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.
Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.
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Tomasz J. Wodzicki
Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.
Attenuation of Endoplasmic Reticulum Stress–Related Myocardial Apoptosis by SERCA2a Gene Delivery in Ischemic Heart Disease
Xin, Wei; Lu, Xiaochun; Li, Xiaoying; Niu, Kun; Cai, Jimei
Previous studies suggested that endoplasmic reticulum (ER) stress–associated apoptosis plays an important role in the pathogenesis of ischemic heart disease. Gene transfer of sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) attenuates myocardial apoptosis in a variety of heart failure models. This study is to investigate the effects of SERCA2a gene delivery on the myocardial apoptosis and ER stress pathway in a porcine ischemic heart disease model. Eighteen pigs were either subjected to a...
GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy.
Holczer, Marianna; Bánhegyi, Gábor; Kapuy, Orsolya
The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress.
Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress.
Li, Shengcun; Zhang, Lulu; Ni, Rui; Cao, Ting; Zheng, Dong; Xiong, Sidong; Greer, Peter A; Fan, Guo-Chang; Peng, Tianqing
Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4weeks) were fed a high fat diet (HFD) or normal diet for 20weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity. Copyright © 2016 Elsevier B.V. All rights reserved.
The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals
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A.J. Parodi
Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.
Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells
Aulestia, Francisco J.; Rodríguez-García, Arancha; Alonso, María Teresa; García-Sancho, Javier
Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating a...
Endoplasmic Reticulum-Associated Degradation Factor ERLIN2: Oncogenic Roles and Molecular Targeting of Breast Cancer
factor-independent proliferation in insulin-like growth factor-deficient media . To further examine the effects of ERLIN2 in a context that more... media . Oncogenesis-associated conditions, such as nutrient or growth factor depletion, can cause pathophysiologic ER stress [22,23]. When MCF10A cells...activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae . Mol Cancer Res 2005; 3
Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum
Echevarría, Wihelma; Leite, M. Fatima; Guerra, Mateus T.; Zipfel, Warren R.; Nathanson, Michael H.
Calcium is a second messenger in virtually all cells and tissues1. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nucle...
Endoplasmic Reticulum Stress: Its Role in Disease and Novel Prospects for Therapy
Schönthal, Axel H.
The endoplasmic reticulum (ER) is a multifunctional organelle required for lipid biosynthesis, calcium storage, and protein folding and processing. A number of physiological and pathological conditions, as well as a variety of pharmacological agents, are able to disturb proper ER function and thereby cause ER stress, which severely impairs protein folding and therefore poses the risk of proteotoxicity. Specific triggers for ER stress include, for example, particular intracellul...
Endoplasmic reticulum stress is induced in the human placenta during labour
Veerbeek, J.H.W.; Tissot Van Patot, M.C.; Burton, G.J.; Yung, H.W.
Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in?vitro. Here, we explored whether exposure to labour represents an in?vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2? and XBP-1, were significantly higher in laboured placentas than in ...
Hesperidin inhibits ovarian cancer cell viability through endoplasmic reticulum stress signaling pathways
Zhao, Jun; Li, Yali; Gao, Jinfang; De, Yinshan
Hesperidin is a vitamin P flavonoid compound primarily present in citrus fruits. The aim of the present study was to investigate whether hesperidin inhibits ovarian cancer cell viability via endoplasmic reticulum stress signaling pathways. A2780 cells were treated with various doses of hesperidin for 6, 12 or 24 h, and the viability of A2780 cells was assessed using the MTT assay. Hesperidin decreased the viability of A2780 cells and increased cytotoxicity in a dose- and time-dependent manner...
Effect of NB-UVB on cytokines and endoplasmic reticulum stress in psoriasis vulgaris lesions
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Xiao-Yan Sun1
Full Text Available Objective: To study the effect of NB-UVB on cytokines and endoplasmic reticulum stress in psoriasis vulgaris lesions. Methods: Patients with psoriasis vulgaris who received NB-UVB therapy in People's Hospital of Beijing Daxing District between May 2014 and January 2017 were selected, proper amount of skin lesion tissue was collected before treatment as well as 4 weeks and 8 weeks after treatment respectively to extract the protein in it, and the protein expression levels of inflammatory cytokines, transcription factors and endoplasmic reticulum stress molecules in tissue protein were determined. Results: 4 weeks and 8 weeks after treatment, IFN-γ, IL-2, IL-17, Runx3 and RORγt expression in lesions were significantly lower than those before treatment while IL-4, IL-10, Foxp3, IRE-1α, XBP1, ATF6, CHOP and GADD34 expression were significantly higher than those before treatment; 8 weeks after treatment, IFN-γ, IL-2, IL-17, Runx3 and RORγt expression in lesions were significantly lower than those 4 weeks after treatment while IL-4, IL-10, Foxp3, IRE-1α, XBP1, ATF6, CHOP and GADD34 expression were significantly higher than those 4 weeks after treatment. Conclusion: NB-UVB can regulate the differentiation and maturation of CD4+ T cell subsets Th1/Th2 and Th17/Treg as well as the apoptosis mediated by endoplasmic reticulum stress in psoriasis vulgaris lesions.
Sarcolipin expression is repressed by endoplasmic reticulum stress in C2C12 myotubes.
Takahashi, Nobuhiko; Kimura, Atsushi P; Naito, Sumiyoshi; Yoshida, Mika; Kumano, Osamu; Suzuki, Takeshi; Itaya, Satoshi; Moriya, Mitsuru; Tsuji, Masahiro; Ieko, Masahiro
Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca2+ ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4μ8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12 myotubes.
Selected terpenoids from medicinal plants modulate endoplasmic reticulum stress in metabolic disorders.
Beukes, Natasha; Levendal, Ruby-Ann; Frost, Carminita L
The majority of research performed on cellular stress and apoptosis focuses on mitochondrial dysfunction; however, the importance of the endoplasmic reticulum dysfunction and the link to metabolic diseases has gained a substantial interest. This review focuses on the potential of terpenoids to influence endoplasmic reticulum stress and the possible role terpenoids play as the treatment of metabolic diseases. Metabolic diseases develop as a result of a cascade of cellular pathways. In most cases, cells are able to compensate for the disruption of the cellular homeostasis although the initiation of response pathways; however, chronic stress initiates apoptotic pathways. This reviewed (1) showed the importance of phytoterpenoids to influence endoplasmic reticulum (ER) stress and homeostasis, (2) showed how regulating ER stress affect the cell survival and death, and (3) highlighted some examples of how the progression of metabolic diseases can be influenced by ER. Due to the substantial number of terpenoids that have been identified in literature, this review gave examples of 21 terpenoids that have been documented to have an effect on the different proteins associated with ER stress, how these plant terpenoids influence ER dysfunction and metabolic diseases such as diabetes, cancer, liver, and neurological diseases and parasitic infections. © 2014 Royal Pharmaceutical Society.
Majumdar, Arun [Orinda, CA; Satyanarayana, Srinath [Berkeley, CA; Yue, Min [Albany, CA
A sensor may include a membrane to deflect in response to a change in surface stress, where a layer on the membrane is to couple one or more probe molecules with the membrane. The membrane may deflect when a target molecule reacts with one or more probe molecules.
Magnetically controlled permeability membranes
Kosel, Jurgen
A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.
Endoplasmic reticulum protein 29 (ERp29, a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse
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Zhu Yemin
Full Text Available Abstract Background Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29, which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process. Methods Laser scanning confocal microscopy (LSCM and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29 to characterize: 1 fertilization rate (FR; 2 fertilization index (FI; 3 sperm motility and 4 acrosome reaction (AR. Results Confocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion antibodies
The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells.
Vera, Carolina Andrea; Oróstica, Lorena; Gabler, Fernando; Ferreira, Arturo; Selman, Alberto; Vega, Margarita; Romero, Carmen Aurora
Ovarian cancer is the seventh most common cancer among women worldwide, causing approximately 120,000 deaths every year. Immunotherapy, designed to boost the body's natural defenses against cancer, appears to be a promising option against ovarian cancer. Calreticulin (CRT) is an endoplasmic reticulum (ER) resident chaperone that, translocated to the cell membrane after ER stress, allows cancer cells to be recognized by the immune system. The nerve growth factor (NGF) is a pro-angiogenic molecule overexpressed in this cancer. In the present study, we aimed to determine weather NGF has an effect in CRT translocation induced by cytotoxic and ER stress. We treated A2780 ovarian cancer cells with NGF, thapsigargin (Tg), an ER stress inducer and mitoxantrone (Mtx), a chemotherapeutic drug; CRT subcellular localization was analyzed by immunofluorescence followed by confocal microscopy. In order to determine NGF effect on Mtx and Tg-induced CRT translocation from the ER to the cell membrane, cells were preincubated with NGF prior to Mtx or Tg treatment and CRT translocation to the cell surface was determined by flow cytometry. In addition, by western blot analyses, we evaluated proteins associated with the CRT translocation pathway, both in A2780 cells and human ovarian samples. We also measured NGF effect on cell apoptosis induced by Mtx. Our results indicate that Mtx and Tg, but not NGF, induce CRT translocation to the cell membrane. NGF, however, inhibited CRT translocation induced by Mtx, while it had no effect on Tg-induced CRT exposure. NGF also diminished cell death induced by Mtx. NGF effect on CRT translocation could have consequences in immunotherapy, potentially lessening the effectiveness of this type of treatment.
F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo.
Berends, Christian W H; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J R; van den Heuvel, Sander
The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by