Substoichiometric inhibition of A\xce\xb21-40 aggregation by a tandem A\xce\xb240-1-Gly8-1-40 peptide
A\xce\xb2 peptides aggregate to form insoluble and neurotoxic fibrils associated with Alzheimer\'s disease. Inhibition of the aggregation has been the subject of numerous studies. Here we describe a novel, substoichiometric inhibitor of A\xce\xb21-40 fibrillization as a tandem dimeric construct consisting of A\xce\xb240-1 (reverse sequence) linked to A\xce\xb21-40 via an eight residue glycine linker. At molar ratios of the tandem peptide to A\xce\xb21-40 of 1:10 to 1:25 inhibition of fibrillization, as measured by Thioflavin T, was observed. We postulate that the tandem construct binds to a fibrillar intermediate but the reverse sequence delays or prevents further monomer association.
1. INTRODUCTION<!>2.1 Preparation of the Tandem peptide and A\xce\xb21-40<!>2.2 Preparation of Peptide Solution and ThioflavinT assay<!>2.3 Atomic Force Microscopy (AFM) of Amyloid fibrils<!>2.4 Circular Dichroism Spectra of Tandem pepitide<!>3.1 The ThioflavinT assay<!>3.2 Atomic force Microscopy<!>3.3 Circular Dichroism spectra of the Tandem peptide<!>4. Discussion<!>5. Conclusion
<p>The aggregation of amyloid-Î² (AÎ²) is believed to be associated with the onset of Alzheimer's disease and there have been numerous studies of compounds that affect the aggregation process. A large number of compounds have been observed to inhibit aggregation and these have generally been grouped into three classes: those that inhibit oligomerization, those that inhibit oligomerization and fibrillization and those that inhibit fibrillization only (1-9). Among these studies, several investigators have used small peptides that would be expected to bind to full length AÎ² but would be unable to form any structure that could serve as a template for subsequent monomer (or oligomer) addition (10,11). These latter studies were based on the observation of Tjernberg et al. (12) which suggested that the central region of the AÎ² peptide (residues16-20) was responsible for the self-association and subsequent aggregation. These inhibitory peptides include those that are N-methylated (13,14) or cross-linked by a disulfide bond (15) or linked to an oligolysine (16). In general, such small peptides are indeed effective in preventing either oligomer formation or aggregation (17, 18) but the concentration of the peptide inhibitors required to prevent aggregation is high suggesting that the binding of the peptide is weak. Recently, Taylor et al. reported a class of retro-peptides which inhibited aggregation of either AÎ²1-40 or AÎ²1-42 at substoichiometric concentrations (19).</p><p>Here we report a novel approach to affect aggregation by using a tandem peptide consisting of two AÎ² molecules linked by a glycine linker in such a way to give the tandem peptide construct NH2-AÎ²40-1-(Gly)8-AÎ²1-40-COOH. In this construct the N-terminal AÎ²40-1 peptide has the reverse sequence of wild-type AÎ² while the C-terminal AÎ² peptide has the forward sequence.</p><!><p>In our studies both AÎ²1-40 and the tandem peptide were prepared as a fusion proteins as recently described by Garai et al. (20). The fusion protein of the tandem peptide was purified from inclusion bodies after dissolving in 6 M guanidine-HCl (GdnHCl), passed over a Ni2+-column and refolded on a Sephacryl S100 column in 25 mM HEPES with 100 mM NaCl at pH 7.5. The fusion protein eluted from the S100 column in two main fractions, one monomeric (60%) and the other multimeric (40%). Both fractions were treated with Factor Xa (New England Biolabs,USA) for 6 hrs at room temperature in 25 mM HEPES, pH 7.5 and 100 mM NaCl, using a ratio of Factor Xa to fusion protein of 1:100 (w/w). After cleavage, the peptide was precipitated with 200 Î¼M ZnCl2, dissolved in 6 M GdnHCl and purified by HPLC. The tandem peptide eluted at 33.5-35% in an acetonitrile gradient, was lyophilized and stored at -20 Â°C. The yield of tandem peptide was 1mg/6L cell culture and showed a typical tyrosine fluorescence spectrum. Mass spectrometry of the purified material showed a single peak at a molecular weight of 9097.64 Da compared to the theoretical expected average isotopic mass of 9098 Da (supplementary figure S1). The peptide concentrations were measured by absorbance using values of 1450 M-1cm-1 and 2900 M-1cm-1 at 276 nm for AÎ²1-40 and the tandem peptide, respectively.</p><!><p>The time course of aggregation in the presence and absence of the tandem peptide was measured by Thioflavin T (Figure1). For these experiments, AÎ²1-40 and the tandem peptide at concentrations of 200 Î¼M and 50 Î¼M respectively were dissolved in 5 mM NaOH. A 50 mM borate stock solution at pH 7.8 was then added to this solution to lower the pH to 8. The final concentration of borate buffer was kept at 25 mM. The solution was centrifuged at 14000g for 20-30 minutes and the supernatant solution was used for the experiments. Varying concentration of AÎ²1-40 and the tandem peptide were incubated at 37 Â°C and the extent of aggregation was measured by Thioflavin T.</p><p>For ThioflavinT assay, 20 Î¼l of the peptide solution was added in 1ml of glycine buffer (50 mM, pH 9.0) containing 10 Î¼M of ThioflavinT. Solutions were excited at 450 nm and emission was recorded at 490 nm. Before each reading a blank containing no peptide solution was taken to check the baseline. Between each reading the cuvettes were thoroughly rinsed with distilled water and dried to avoid any contamination.</p><!><p>For AFM studies, 75 Î¼M AÎ²1-40 in the presence and absence of 3 Î¼M tandem peptide was incubated for 4 days at 37 Â°C in 25 mM borate buffer at pH 8.0. 5Î¼L of sample was diluted 20-fold with distilled water and allowed to settle on the freshly cleaved mica surface for 30 min. The majority of the solution was drawn off and the samples dried for 10-15 minutes in vacuo. The images were recorded using AFM MFP-3D-BIO (Asylum Research, Santa Barbara) with a scan resolution of 512Ã512 points/scan at a scan speed of 1 Hz.</p><!><p>For secondary structural analysis with CD, a tandem peptide stock solution (50 Î¼M) in 5 mM NaOH solution buffer was diluted 5-fold into 200 mM phosphate buffer at pH 8.0.The CD scans were recorded immediately after dilution using a Jasco J-715 spectropolarimeter. Ten scans from 200 to 260 nm with speed of 20 nm/minute were averaged. A buffer blank was performed under the same conditions and the final spectra were analyzed after buffer blank subtraction.</p><!><p>Figure 1A shows that the onset of fibrillization in the presence of the tandem peptide at a tandem pepide:AÎ²1-40 molar ratio of 1:25 is delayed about 2-fold. At a tandem peptide:AÎ²1-40 molar ratio of 1:10 (Figure 1B), no fluorescence increase is observed after 350 hrs compared to the fibrillization being complete by 175 hrs in the absence of the tandem peptide. The concentrations of AÎ²1-40 used in these experiments are 50 Î¼M and 65 Î¼M respectively, while those of tandem peptides are 2 and 6.5 Î¼M, respectively. As a control experiment, two reference samples containing 2 Î¼M and 6.5 Î¼M of tandem peptide alone were kept under the same experimental conditions. Neither increase in ThioflavinT signal nor any precipitation was observed even after 12 days of incubation.</p><!><p>Figure 2 shows AFM images of AÎ²1-40 in the absence and presence of tandem peptide at a 1:25 ratio. Figure 2A clearly shows large fibrils (~20 Î¼m in length) in the absence of the tandem peptide after 4 days of incubation while no fibril formation was observed in the presence of the tandem peptide (Figure 2B).</p><!><p>The CD spectrum of the tandem peptide showed a characteristic minimum at 216 nm suggesting a high Î²-sheet propensity (Figure 3). Interestingly, when the tandem peptide was kept for 4-5 days under these conditions at concentration ~10 Î¼M no increase in ThioflavinT signal was observed.</p><!><p>The experimental results clearly indicate a sub-stoichoimeric inhibition of AÎ²1-40 fibrilization by the tandem peptide. The present study represents a different and unique approach to study the aggregation mechanism of AÎ². The results presented here not only suggest a mechanism for AÎ²1-40 aggregation but also a general procedure for inhibiting other aggregation processes. The substoichiometric inhibition suggests first that the tandem peptide is binding to a form, presumably an oligomer, which is on pathway to form fibrils, and which is present in only low concentration. From the CD data, the tandem peptide shows a high Î²-sheet propensity which might allow specific binding to an intermediate directly involved in fibrillization pathway. On the other hand, it is known that reverse sequence AÎ²40-1 has different properties than that of normal AÎ²1-40. For example, Giordano et. al., have suggested that both the nature of aggregates formed and the mechanism of toxicity differs between the peptides containing the forward and reverse sequences (21). The reverse sequence AÎ²40-1 forms globular aggregates in contrast to the forward sequence which is fibrilar (21). This is indeed what we see in AFM when the tandem peptide aggregates at higher concentration (â¥ 25Î¼M) (supplementary figure S2).</p><p>Jan et al. (22) have shown that while AÎ²1-40 appears to retard the formation of mature AÎ²1-42 fibrils, the reverse sequence AÎ²40-1 does not affect fibrillization of protofibrillar AÎ²1-42 indicating the specificity of the interaction. Thus in the complex formed by tandem peptide and the on pathway oligomer(s) involved in fibrilization, the reverse sequence present in the tandem peptide may not allow monomer addition thereby preventing further aggregation.</p><!><p>These studies suggest a new approach for understanding the mechanism of fibrillization of intrinsically disordered proteins. Such studies are first of its kind and various permutation of such sequence attachment in AÎ² is presently being pursued in our lab to check their effects on fibrilization. We are also currently in process of implementing tandem constructs in vivo to check its effect on aggregation of AÎ² inside neuronal cells.</p>