A specific fluorescent probe reveals compromised activity of methionine sulfoxide reductases in Parkinson's disease
Oxidation of methionine residues to methionine sulfoxide (MetSO) may cause changes in protein structure and function, and may eventually lead to cell damage. Methionine sulfoxide reductases (Msrs) are the only known enzymes that catalyze the reduction of MetSO back to methionine by taking reducing equivalents from the thioredoxin system, and thus protect cells from oxidative damage. Nonetheless, a lack of convenient assays for the enzymes hampers the exploration of their functions. We report the discovery of Msr-blue, the first turn-on fluorescent probe for Msr with a >100-fold fluorescence increment from screening a rationally-designed small library. Intensive studies demonstrated the specific reduction of Msr-blue by the enzymes. Msr-blue is ready to determine Msr activity in biological samples and live cells.Importantly, we disclosed a decline of Msr activity in a Parkinson's model, thus providing a mechanistic linkage between the loss of function of Msrs and the development of neurodegeneration. The strategy for the discovery of Msr-blue would also provide guidance for developing novel probes with longer excitation/emission wavelengths and specific probes for Msr isoforms.
Introduction<!>Probe library synthesis<!>Probe screening<!>Probe validation<!>Specic activation of Msr-blue by Msrs<!>Conclusions
<p>Methionine side chains in proteins/peptides are among the most susceptible to oxidation by reactive oxygen/nitrogen species, leading to the formation of methionine sulfoxide (MetSO) residues. Methionine sulfoxide reductases (Msrs), the only characterized enzymes known to reduce the MetSO residues in oxidatively damaged proteins/peptides to methionine residues by taking reducing equivalents from the thioredoxin system, are virtually universal among all aerobic organisms. [1][2][3][4][5] Msr A and Msr B are two major isoforms in mammals and specically reduce the S-MetSO epimer and R-MetSO epimer, respectively. 6,7 The structure of the Msr-substrate complex shows that the methyl sulfoxide moiety ts in the surfaceexposed active site of the enzyme, where the oxygen of the sulfoxide forms an intensive hydrogen bonding network with several polar residues. However, the other parts of the substrate are exible. [8][9][10] Thus, Msrs have broad substrate specicity and can reduce a variety of methyl sulfoxide-containing molecules, ranging from protein bound MetSO residues to several small molecules, such as dimethyl sulfoxide, methionine sulfoxide, sulindac and sulforaphane. [11][12][13] Despite the important roles of Msrs in regulating multiple cellular events, 1,4,14,15 the lack of convenient tools to detect their activity hampers the exploration of the function of the enzymes under physiological or pathophysiological conditions.</p><p>Developing specic probes for a particular biomolecule is of great importance to unveil its function in physiological/pathophysiological processes. Among various detection techniques, uorescence-based assays are widely adopted due to their high sensitivity, operational simplicity, and super biocompatibility. Therefore, the past years have witnessed an increase in the number of studies that focus on developing diverse uorescent probes for studying various protein activities [16][17][18][19][20][21][22][23][24][25][26][27] as well as small biomolecule functions. [28][29][30][31][32][33][34][35] One of the outstanding and wellrecognized strategies in designing uorescent probes is the manipulation of the intramolecular charge transfer (ICT) process, as the alteration of such a process is usually accompanied with a pronounced change in the absorbance/uorescence proles of a molecule, which forms the basis of the uorescence sensing. Manipulation of the ICT process could be easily achieved by tuning the electronic character of the substituents attached to a uorophore by exchanging electron donating groups with electron withdrawing groups or vice versa. 36,37 The reaction that is catalyzed by Msr converts sulfoxides to the corresponding suldes, which enables the transformation of an electron withdrawing group to an electron donating group. Taking into consideration the broad substrate scope of Msrs, we thus hypothesized that attachment of the methyl sulfoxide moiety to certain uorophores might generate turn-on probes for the enzymes (Scheme 1).</p><p>We created a focused small library containing 23 dyes uniformly decorated with a methyl sulfoxide group (1-23, Fig. 1), and evaluated their reduction by Msr. The synthetic routes are illustrated in the ESI † (Schemes S1-3). Characterizations and the original spectra ( 1 H NMR, 13 C NMR, and MS) of the nal compounds are included in the ESI † (Fig. S4-71). Compound 15 (Msr-blue) was identied as the rst turn-on uorescent probe for Msr with a more than 100-fold uorescence increment. The specic activation of Msr-blue by Msr was further demonstrated. Msr-blue is ready to image enzyme function in live cells and to measure enzyme activity in biological samples. With the aid of Msr-blue, a decline in enzyme activity in a cellular model of Parkinson's disease (PD) was disclosed for the rst time. We expect the probe would facilitate an understanding of the physiological/pathophysiological functions of Msrs. In addition, the concept of the probe design would also advance the development of novel Msr probes with longer excitation/emission wavelengths as well as specic Msr A and Msr B probes.</p><!><p>Diverse uorophores, such as naphthalimide, BODIPY, cyanine and coumarin, were selected to construct the target probe library. A uniform methyl sulfoxide group was introduced by oxidation of the corresponding precursor methyl suldes under mild conditions to furnish the nal 23 potential probes (Fig. 1). The straightforward synthetic routes are illustrated in the ESI † (Schemes S1-3). All of the nal 23 compounds were fully characterized by MS and NMR, and their original spectra ( 1 H NMR, 13 C NMR, and MS) are included in the ESI † (Fig. S4-71). The purity of the probes 15 and 16 was determined to be 98% and 99%, respectively (Fig. S72 †).</p><!><p>Aer constructing a small library of methyl sulfoxide dyes, we next screened the library for potential Msr uorogenic substrates (Table 1). As we employed DTT as the electron donor Scheme 1 Design of potential probes for Msrs.</p><p>Fig. 1 The sulfoxides library.</p><p>for the reduction of sulfoxides under Msr catalysis, we thus rstly investigated whether DTT causes interference in the assay. As shown in Table 1, uorescence signals of ve compounds (1, 2, 3, 14 and 17) were efficiently activated by DTT (F/F 0 > 10). This is not surprising as sulfoxides are prone to be reduced by thiols. 36,38 In addition, compounds 10 and 22 and their corresponding suldes (10 0 and 22 0 ) have very weak uorescence in aqueous solutions. Consequently, these seven compounds were excluded in the next studies using both DTT and Msr A. To our delight, two (15 and 16) out of the sixteen DTT-negative compounds showed a 32.3-and 11.6-fold increase in their uorescence signal, respectively. We therefore picked up compounds 15 and 16 for the follow-up validation studies.</p><!><p>Firstly, we performed experiments to conrm the response of both probes to DTT by using varying concentrations of the reductant. As shown in Fig. 2A, high concentrations of DTT cause a signicant elevation in the uorescence signal of 16, while DTT appears to affect 15 much less. In addition, compound 16 shows only about a maximum 10-fold increase in emission under Msr A catalysis (Fig. S1 †). Considering the potential interference of DTT with 16 and the lower uorescence increase of 16 under enzyme catalysis, we thus decided to focus on the probe 15 in the following experiments. The DTT  concentration was xed at 5 mM in the following assays, as DTT at this concentration causes negligible interference in the uorescence signal. Since the probe 15 emits blue uorescence aer activation by Msr A, we termed it as Msr-blue. Msr-blue responded to Msr A in both a time-and dose-dependent manner (Fig. 2B and C), and more than a 100-fold increase in the emission was observed. HPLC analysis of the reaction demonstrated that Msr-blue was converted to its corresponding sulde (15 0 ) under catalysis by either the puried Msr A or a cell lysate (Fig. 2D and S2 †). When incubation of Msr-blue (10 mM) with DTT and Msr A in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.4) took place for 6 h at 37 C, the desired sulde 15 0 was found in the reaction mixture with $40% conversion. At the time point of analysis, the remaining amount of Msr-blue (6.2 mM) plus the product 15 0 (3.8 mM) was equal to the amount of the starting Msr-blue (10 mM), indicating that the probe was exclusively converted to the expected sulde 15 0 . It should be noted that Msr-blue is a racemic mixture containing both S-epimers and Repimers, and Msr A should only convert the S-epimer to the sulde 15 0 . Incubation of Msr-blue with mouse kidney lysate gave similar results as those for the puried enzyme, yet with a lower conversion rate of the probe ($14% conversion, Fig. S2 †). The sulde 15 0 and the reaction mixtures which were catalyzed by puried Msr A and by the cell lysate gave similar uorescence proles (Fig. 2E), supporting the conversion of Msr-blue to the sulde 15 0 under Msr catalysis. Taken together, we rmly concluded herein that the probe Msr-blue could be reduced by Msr to the corresponding sulde, leading to a more than 100-fold increase in the uorescence signal.</p><p>To further demonstrate the specic reduction of one epimer in the racemic mixture of Msr-blue, we further compared the reduction of the probe by the pure Msr A enzyme and the mouse kidney lysate, which contains both Msr A and Msr B enzymes. As shown in Fig. 2F, the reaction catalyzed by Msr A could be nished within 6 h, and the maximal formation of the sulde was less than 50% ($42% aer 8 h). In contrast, the kidney extract reduces more Msr-blue: the yield of the corresponding sulde is >60% at 8 h, and could generate >70% of the sulde aer incubating the probe for 24 h. Aer the Msr A-catalyzed reaction was complete, we added the kidney extract to the reaction mixture. Again, formation of the sulde continues (Fig. 2G), and $80% total sulde was generated aer incubating the mixture with the lysate for an additional 8 h. Our results support the conclusion that Msr A could specically reduce one epimer (likely the Sisoform) of Msr-blue, and that preparation of enantiomers of Msr-blue would yield specic probes for Msr A/B enzymes.</p><!><p>Next, we performed experiments to answer whether the uorescence signal of Msr-blue could be specically turned on by Msrs. Firstly, the response of Msr-blue to a panel of potential reducing agents and enzymes was tested (Fig. 3A). Under our experimental conditions, Msr A turns on the uorescence with nearly 80-fold increase, while none of the tested species, such as various small molecule thiols, glutathione reductase (GR), thioredoxin (Trx), thioredoxin reductase (TrxR), bovine serum albumin (BSA), ascorbate, and tris(2-carboxyethyl)phosphine (TCEP), turn on the uorescence. We then measured the reduction of Msr-blue in the presence of the Msr inhibitors dimethyl sulfoxide (DMSO) and MetSO. As shown in Fig. 3B-D, both inhibitors suppressed the catalytic reduction of Msr-blue by either the puried enzyme or the protein extract from mouse kidneys. We further examined the reduction of the probe by live cells. As shown in Fig. 3E, the live 293T cells have a weak background signal. Aer incubation with Msr-blue, the blue uorescence signal increases time-dependently, and it appears to reach saturation aer incubation for 4 h. Again, the uorescence signal could be inhibited by pretreatment of the cells with DMSO. Imaging Msr activity in HL-60 cells and its inhibition by DMSO was also demonstrated (Fig. S3 †). Next, we applied the probe to measure the Msr activity in different mouse organs, i.e. liver, spleen, kidney, brain and heart. As shown in Fig. 4A, the kidney appears to show the highest activity, followed by the liver, and then the brain. The heart and spleen show the lowest activity. Coincidentally, the protein level of Msr A follows the same trend: kidney > liver [ brain > heart $ spleen, which matches well with the results from the Msr-blue-based uorescence assay (Fig. 4C). To further demonstrate the specic reduction of Msr-blue by Msr, we compared the reduction of the probe by mouse kidney lysates before and aer an immunodepletion of Msr A. The depletion of Msr A from the lysate remarkably decreased the uorescence intensity ($25% decline, Fig. 4B). The efficiency of the immunoprecipitation (IP) was validated by Western blots (Fig. 4D). It was estimated by densitometry using ImageJ that more than 60% of Msr A was removed from the lysate. The Msr-blue reduction activity in the lysate aer IP is likely from both Msr B and the remaining Msr A, since the Msr A antibody does not crossreact with Msr B. Taken together, we concluded herein that Msr-blue is a specic uorogenic substrate of Msrs. The kinetics of the Msr A-catalyzed Msr-blue reduction indicated that Msr-blue is a good substrate for Msr A with a K m of 120 mM and a k cat of 0.4 s À1 (Fig. S73 †), which are similar to the values of other MetSOcontaining substrates. 15,39 Decline of Msr activity in a PD model Due to the critical roles of Msrs in defending against oxidative stress and preventing accumulation of misfolded proteins, a compromised function of Msr has been linked to the development of aging-associated disorders like neurodegenerative diseases. 4,[40][41][42] In line with this observation, an overexpression of Msr A confers benets against the development of PD, 40,41,43 while knockout of the enzyme enhances the progression of neurodegeneration. 42 Markesbery et al. reported the decrease of Msr A activity in brains that have been affected by Alzheimer's disease. 44 However the activity of Msrs in PD tissues or models is unknown. 6-Hydroxydopamine (6-OHDA) is a well-established neurotoxin used to generate PD models in animals or cells. 45,46 We employed the 6-OHDA-treated PC 12 cells as a cellular model of PD [47][48][49] and applied Msr-blue to probe the function of Msrs in the cells. As illustrated in Fig. 5A, 6-OHDA treatment causes a remarkable decline in the enzyme activity, revealed by the decrease in the uorescence intensity of the cells. As the activity of Msrs is supported by the thioredoxin system in cells, we thus further determined their activity in the lysate by supplying DTT as a reductant to exclude the possible alteration of function by the thioredoxin system. Consistent with the Msrblue imaging results, the activity of Msr decreased by $30% in the drug-treated cells compared to the control groups (Fig. 5B). Analysis of the protein levels of Msr A indicated no signicant alteration of the protein expression aer 50 mM of 6-OHDA treatment, yet a slight decrease aer 100 mM of 6-OHDA treatment (Fig. 5C). Taken together, with the aid of Msr-blue, a decline of the Msr activity in a PD model was disclosed for the rst time.</p><p>The methionine side chain is highly susceptible to oxidation particularly under conditions of oxidative stress. Oxidation of methionine residues to MetSO, usually leading to a change of protein structure and alteration of protein function, could be reversed by taking reducing equivalents from the thioredoxin system catalyzed by Msr. Via catalyzing regeneration of methionine from MetSO, Msr regulates the function of a diverse variety of proteins, ranging from protein kinases to transcription factors and metabolic enzymes. 1,4,15 However, the lack of convenient methods to assay the enzyme activity hampers the exploration of their functions. During preparation of the manuscript, we noticed that Dr Makukhin et al. reported the rst ratiometric uorescent probe, Sulfox-1, for Msr A. 50 In this paper, the authors used a chiral starting material to construct the optical pure probe (S)-Sulfox-1, which guided us to prepare (S)-or (R)-epimers of the probe for selective Msr A and Msr B imaging. However, compared to Msr-blue, there are some weak  points of Sulfox-1. Firstly, the authors demonstrated that Msr A could reduce the probe, giving a $7-fold decrease in emission at 542 nm and a very slight increase (less than 1.5-fold) in emission at 576 nm. In contrast, Msr-blue is almost non-uorescent, and displays a >100-fold increase in uorescence upon responding to Msr A. This off-on character and large magnitude elevation of the uorescence signal provide the easiest way to measure the enzyme activity. Secondly, the application of Sulfox-1 is quite limited: the authors only performed live cell imaging using bacterial cells. The practical application of Msrblue was demonstrated by various experiments. This probe is suitable to image Msr function in different types of mammalian cells and assay the enzyme activity in biological samples. More importantly, Msr-blue was used to probe the activity of Msr in a PD model, which reveals a remarkable decline in the enzyme activity in this disease for the rst time. Thirdly (and most importantly), the specicity of Sulfox-1 for the enzyme is not well addressed as the paper lacks such data. It's well known that sulfoxides are prone to be reduced by thiols. Indeed, the authors found that DTT could reduce Sulfox-1. We performed intensive research to demonstrate that Msr-blue is activated by Msr specically, which guarantees its application to explore the physiological/pathological function of Msrs. The disadvantage of Msr-blue is its short excitation/emission wavelengths. Based on the strategy described in this paper, we are currently preparing novel probes by conjugation of the methyl sulfoxide group to more uorophores with green or red emission, and expect to discover specic probes with longer excitation/emission wavelengths.</p><!><p>In summary, we have rationally designed a focused sulfoxide library, and identied Msr-blue as the rst specic off-on probe of Msr with a >100-fold increase in the emission. The probe is ready to use and multiple practical applications of the probe have been demonstrated. With the aid of Msr-blue, we disclosed the compromised Msr activity in a PD model, highlighting the relevance of Msr dysfunction to the progress of neurodegeneration. We expect that Msr-blue would be a powerful tool to facilitate dissecting the functions of Msrs. In addition, the strategy for the discovery of Msr-blue also sheds light on the development of Msr probes with longer excitation/emission wavelengths as well as specic Msr A or Msr B probes.</p>