Acceleration of α-synuclein aggregation by homologous peptides

FEBS Letters 580 (2006) 3657–3664

Acceleration of a-synuclein aggregation by homologous peptides Hai-Ning Dua, Hong-Tao Lia, Feng Zhangb, Xiao-Jing Lina, Jia-Hao Shia, Yan-Hong Shic, Li-Na Jib, Jun Hub, Dong-Hai Linc, Hong-Yu Hua,* a

Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China b Shanghai Institute of Applied Physics, Shanghai 201800, China c Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Shanghai 201203, China Received 22 March 2006; revised 18 May 2006; accepted 19 May 2006 Available online 2 June 2006 Edited by Judit Ova´di

Abstract a-Synuclein (a-Syn), amyloid b-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66–74) on a-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type a-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient a-Syn mutant. The acceleration of a-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in a-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: a-Synuclein; GAV motif; Aggregation; Fibrillization; Homologous peptide; Parkinson’s disease

1. Introduction It is generally considered that protein amyloidogenesis or fibrillogenesis is closely associated with many neurodegenerative diseases [1,2], such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and prion disease. One pathological hallmark of these disorders is the deposition of abnormal fibrillar proteins or peptides in senile plaques or inclusion bodies in patient brains [3]. a-Synuclein (a-Syn) [4], amyloid b-protein (Ab) [5] and prion protein (PrP) [6] are the three most well studied proteins that are prone to aggregation or even fibrillization both in vivo and in vitro. Sequence alignment indicated that a-Syn, Ab and PrP share a homologous hydrophobic region [7,8], normally with a consensus sequence of VGGAVVAGV (Fig. 1A). We named it the GAV motif [9], as it is mainly comprised of Gly, Ala and Val residues. Several studies have implicated the conserved sequence in the fibrillization and cytotoxicity of the neurodegen* Corresponding author. Fax: +86 21 54921011. E-mail address: [email protected] (H.-Y. Hu).

eration-related proteins. For example, a PrP region (residues 106–126) was proposed to be crucial for prion conversion [10], and a synthetic peptide corresponding to the sequence is highly amyloidogenic and cytotoxic [11]. Some studies concentrated on the central sequence of Ab peptide, suggesting that Ab14–23 [12] and Ab16–22 [13] are the fibril-generating peptide fragments, other study indicated that the Ab36–42 fragment could form very stable fibril with highly ordered structure and is a key factor determining the aggregation of Ab1–42 [14]. This renders us to reconsider why only Ab40 and Ab42 peptides are neurotoxic and why Ab42 aggregates into fibrils faster than Ab40 [5]. a-Syn is another amyloidogenic protein of great interest that is closely associated with the Lewy bodies in Parkinson disease [15,16]. Non-amyloid component (NAC), the central region of a-Syn (residues 61–95), can effectively form amyloid-like fibrils in vitro [17,18] and the aggregated core is resistant to proteolysis [19]. The N-terminus of NAC (residues 8–16) has been shown to be the critical core responsible for aggregation and cytotoxicity [20]. These peptide fragments readily assemble into fibrils with large content of b-sheet structures [8]. On the contrary, the C-terminal fragment of NAC tends to form a-helix structure and likely loses the ability to aggregate in vitro [8,20]. After studying the conserved sequence by mutagenesis combined with biochemical characterization, we previously ascertained that the GAV sequence (66–74) is essential to a-Syn fibrillization and its cytotoxicity [21]. A growing body of evidence supports the idea that there is a core region that may play pivotal roles in initiating the aggregation processes of Ab peptides, a-Syn, the N-terminal flexible segment of PrP and other amyloidogenic proteins [22]. To further study the amyloidogenic cores that may have potential application to drug discovery, we chemically synthesized several 9-residue homologous peptides corresponding to the sequence of residues 66–74 of a-Syn and biochemically characterized their abilities to influence a-Syn aggregation. Our results show that these short peptides can interact with a-Syn and accelerate its fibrillization. This will provide further understanding of the peptide-chain interactions that drive protein fibrillization and assist in the design of accelerator or inhibitor for interfering with the amyloidogenic processes. 2. Materials and methods 2.1. Peptide synthesis Three peptides, namely TGV, GAV and GAR (sequences see Fig. 1), were synthesized by the Boc solid-phase method on a peptide synthesizer and released from the resin by hydrogen fluoride cleavage. The

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.05.050

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A

Aβ 36-44

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PrP117-125

AAGAVVGGL

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Fig. 1. Acceleration of a-Syn aggregation by the homologous peptides detected by turbidity. (A) Sequence alignment of the homologous GAV region from Ab, PrP and a-Syn. The sequence of the TGV peptide corresponds to the residues of 66–74 of a-Syn, while that of the GAV peptide is the consensus of the GAV region. The GAR peptide is designed for a control, which has two Val residues at positions 5 and 6, respectively, substituted with Arg. The –NH2 in the C-termini denotes that these peptides are in the amide forms. (B) Incubation of WT a-Syn or D66–74 mutant (2.25 mg/mL) with different concentrations of the GAV peptide (0.3 mg/mL or 0.6 mg/mL). The data were obtained by analyzing the turbidities of the samples at 330 nm after incubation of 24 h. The fresh WT a-Syn was as a control. (C) As in (B), incubation of WT a-Syn (2.25 mg/mL) with the TGV or GAR peptide (0.3 mg/mL or 0.6 mg/mL). The numerals in the parentheses represent the concentrations of the proteins or peptides (mg/mL). Data shown are the average of triplicate incubations (means ± S.E.). peptides were then purified by using a TSK-40 (S) column (2.0 · 98 cm). The purity of the peptides was estimated to be greater than 99% as evidenced by reverse-phase HPLC on a C8 analytical column (4.6 · 250 mm, Beckman) and amino acid analysis. The peptides were further verified by ESI-MS analysis. The molecular weights of TGV, GAV and GAR are 756.6, 727.6 and 841.6 Da, respectively, which are in good agreement with the predicted (757.9 Da for TGV, 727.9 Da for GAV, 841.9 for GAR). 2.2. Circular dichroism measurement CD spectra of the peptide samples were recorded over the wavelength range 190–250 nm on a Jasco-715 spectropolarimeter. The peptide samples were diluted to a concentration of 0.2 mg/mL in a PBS buffer (10 mM phosphate, 10 mM NaCl, pH 7.0, with 0.05% sodium azide) and transferred into a 1-mm quartz cell. All spectra were corrected by subtracting the baseline of the buffer alone. The parameters for recording CD spectra were used as previously reported [23]. Each spectrum was processed by averaging three scans of the sample and the data were presented as mean residual molar ellipticities (deg cm2/dmol). 2.3. Experiments for peptide-induced aggregation Recombinant human a-Syn and its mutants (G68R, G68E and D66–74) were prepared as previously described [21,24]. In the peptide-induced aggregation experiments, different amount of the fresh peptide was added into the fresh samples of various a-Syn mutants at a concentration of 2.25 mg/mL in a PBS buffer (100 mM phosphate, 100 mM NaCl, pH 7.0, 0.05% sodium azide). The mixtures were incubated in 1.5 mL sterile tubes with continuous shaking at 37 C. The time course of aggregation was monitored by measuring

the turbidity (OD330) [5] and Thioflavin T (ThT, Aldrich) fluorescence [25]. Fluorescence measurements were performed on a Hitachi F-4010 fluorophotometer. At an excitation wavelength of 446 nm, the emission intensities at 482 nm were recorded immediately after addition of ThT solution [24]. The measurements were carried out in triplicate for each sample and the error bars were presented (means ± S.E.). 2.4. Centrifugal sedimentation analysis Non-induced and induced a-Syn proteins with a concentration of 2.25 mg/mL were incubated in PBS buffer at 37 C for 1–2 days with continuous shaking. Samples were ultracentrifuged at 100 000 · g for 20 min. Then the supernatant (S) and pellet (P) were loaded on 15% polyacrylamide gels for electrophoresis followed by Coomassie Blue staining. 2.5. Atomic force microscopy The manipulation was as same as the preceding paper [21]. Briefly, each sample was prepared by displaying 5 lL solution on freshly cleaved mica. Images were obtained with a commercial AFM facility (Nanoscope IIIa, Digital Instruments, Santa Barbara, CA) equipped with a 130 lm · 130 lm scanner (J-scanner) by tapping mode imaging. In order to obtain real height of the fibrils, a minimal force between the tip and the sample was used. Each sample was observed in at least five regions to reduce experimental error. Normally, scanning parameters was varied with individual tip and sample. The heights of the filaments were estimated by section analysis with a software attached in the facility. The morphological data for the fibril species were obtained by averaging a large number of individual filaments (n > 25) in at least three individual imaging experiments (mean ± S.E.).

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Fig. 2. The effects of the GAV peptide on the aggregation of a-Syn and its mutants. (A) Peptide-promoted aggregation of a-Syn protein as monitored by ThT binding assay. Square, WT a-Syn only; open circle, a-Syn with 0.3 mg/mL GAV peptide; solid circle, a-Syn with 0.6 mg/mL GAV peptide; open star, a-Syn with 0.3 mg/mL GAR peptide; solid star, a-Syn with 0.6 mg/mL GAR peptide. The free GAV peptide with concentrations of 0.6 mg/ mL (solid triangle) and 1.2 mg/mL (open triangle) were as the controls. (B) Peptide-induced aggregation of a-Syn mutants as monitored by ThT binding assay. Solid star, D66–74 only; open star, D66–74 with 0.6 mg/mL peptide; solid diamond, G68E only; open diamond, G68E with 0.6 mg/mL peptide; solid triangle, G68R only; open triangle, G68R with 0.6 mg/mL peptide. The numerals in the parentheses represent the concentrations of the proteins or peptides (mg/mL). The concentration of a-Syn and the mutants for the above experiments was 2.25 mg/mL. (C) CD spectra of the aggregates from the G68R mutant. Circle, G68R only for 15-day incubation; triangle, G68R with the GAV peptide for 6-day incubation. (D) CD spectra of the aggregates from the G68E mutant. Circle, G68E only for 15-day incubation; triangle, G68E with the GAV peptide for 15-day incubation. The fresh G68R and G68E samples (square) were as the controls. 2.6. NMR experiments The pET3a vector harboring G68E mutant gene was transformed into E. coli BL21 (DE3). The 15N-labeled protein was expressed in M9 minimal medium containing 15NH4Cl and purified as described in the previous report [26]. All NMR experiments were performed on a Varian Unity Inova 600 MHz spectrometer at 25 C. 15N-labeled G68E protein (200 lM) in a buffer (100 mM phosphate, pH 6.5) with 8% D2O was mixed with the GAV peptide (1:2 or 1:10 molar ratio) and incubated at 37 C for several days. The 1H–15N HSQC spectra were recorded at incubation intervals. The spectra of the peptide-free samples before and after 7-day incubation were also obtained as the controls.

3. Results 3.1. Acceleration of a-Syn aggregation by the homologous peptides To further understand the role of the GAV motif in a-Syn fibrillization [21], we studied several homologous peptides (Fig. 1A). The C-termini of the peptides were capped as amide to reduce peptide charge and flexibility. As shown in Fig. 1, both the GAV and TGV peptides can accelerate the

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aggregation of WT a-Syn based on turbidity detection. On the contrary, the control peptide GAR, in which two central valines are replaced by the arginine residues, slightly reduces the aggregation. By means of ThT fluorescence assay, a-Syn exhibits a faster aggregation rate in the presence of the GAV peptide with a shorter lag phase of 8–12 h and a slightly increased amount of the aggregates as compared with the GAV-free sample (Fig. 2A). Intriguingly, the GAV peptide itself does not show fluorescence enhancement even at a high concentration (1.2 mg/mL), suggesting that the increase of ThT fluorescence intensity at 482 nm originates from the amyloid formation of a-Syn rather than from the peptide selfaggregation. As a comparison, the GAR peptide significantly slows down a-Syn aggregation with a dose-dependent manner. These results demonstrate that the acceleration of a-Syn aggregation by the homologous peptides is sequence specific. We also utilized sedimentation analysis combined with electrophoresis to examine the role of the two peptides in a-Syn aggregation. Before incubation, all WT a-Syn molecules are in the soluble fraction. After incubation of 1–2 days, the majority of a-Syn molecules are in the pellets in the presence of the GAV or TGV peptide (Fig. 3A and B), whereas a considerable amount of a-Syn is retained in soluble fractions in the absence of the peptides. 3.2. Peptide-induced aggregation of the charge-incorporated mutants of a-Syn Our previous study [21] showed that introduction of charged residues into the GAV motif region (G68R, G68E, etc.) could reduce fibril formation of a-Syn; and the G68E mutant is much less potent to form oligomers or short fibrils than G68R. To realize whether the GAV peptide promotes fibrillization of these mutants, we examined the aggregation behavior of the G68R and G68E in the presence of the GAV peptide. As expected, aggregation of G68R is dramatically accelerated by the peptide as evidenced by the significant shortening of the lag phase of the aggregation profile (Fig. 2B). Another mutant,

H.-N. Du et al. / FEBS Letters 580 (2006) 3657–3664

G68E, shows only moderately increased aggregation in the presence of the GAV peptide. CD studies reveal that, with the assistance of the GAV peptide, G68R undergoes structural transformation from random coil to b-sheet (Fig. 2C). In contrast, incubation of G68E with the GAV peptide causes no significant change in far-UV CD spectra except a slight increase of the ellipticity at around 220 nm (Fig. 2D). As shown in Fig. 3C, G68R forms a major part of the aggregate deposits in the presence of the GAV peptide, whereas the peptide-free sample forms no aggregates even after incubation of 6 days. These data indicate that the GAV peptide induces aggregation of G68R more effectively than that of the G68E mutant. 3.3. Peptide-induced fibrillization of G68R and G68E mutants: AFM evidence We further compared the AFM images of the a-Syn fibrils generated in the absence or presence of the GAV peptide. The images demonstrate that, after incubation of 6 days, the peptide induces G68R to form abundant filaments with height of 5.8 nm and length of over 200 nm (Fig. 4B). Similarly, the peptide can also induce G68E to form small short filaments of 6.2 nm in height and around 50 nm in length, but the majorities are oligomers or anomalous aggregates (Fig. 4D). Nevertheless, no fibrils can be visualized in the incubations in the absence of the GAV peptide even after extending the incubation time to 15 days (Fig. 4A and C) as previous reported [21]. 3.4. Peptide-induced conformational change of G68E mutant The above results show that the GAV peptide can induce the fibrillization of charge-incorporated a-Syn mutants. To further understand the mechanism of the peptide-induced fibrillization, we studied G68E, a less potent aggregation mutant, by 1 H–15N HSQC NMR experiments. The chemical shifts of the amides in fresh G68E exhibit small dispersion in the 1H dimension (Fig. 5A), suggesting that G68E is a natively unstructured protein similar to WT a-Syn [26]. Incubation of G68E for 7 days has no significant effect on the HSQC spectrum

Fig. 3. SDS–PAGE for the peptide-induced aggregation of a-Syn and its mutants. (A) a-Syn with the GAV peptide. WT a-Syn was incubated with the GAV peptide: 0.3 mg/mL (the third panel from left), 0.6 mg/mL peptide (the fourth panel) or without the peptide (the second panel). The fresh sample was as a control. S and P denote the supernatant and pellet, respectively. (B) a-Syn with the TGV peptide. WT a-Syn was incubated with the TGV peptide: 0.3 mg/mL (the third panel from left), 0.6 mg/mL peptide (the fourth panel) or without the peptide (the second panel). (C) G68R and (D) D66–74 were incubated for 2 or 6 days with or without the GAV peptide as indicated in the graphs. The major 19-kDa band in a lane is a-Syn protein, and the minor bands with smaller molecular weights are probably a-Syn fragments caused by degradation during incubation.

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(Fig. 5B), suggesting little structural perturbation occurred during the incubation. This is consistent with the previous result that G68E cannot self-aggregate into mature fibrils [21]. When the spectra are acquired immediately after mixing G68E protein with the GAV peptide, no significant chemical-shift change occurs (Fig. 5C and E), suggesting that the peptide does not rapidly associate with the a-Syn mutant upon mixing. However, after the mixture is incubated for 7 days, large chemical-shift changes occur in the spectra (Fig. 5D and F). Similar results are also obtained with a shorter incubation of 3 days (data not shown). The higher concentration of the peptide seems to cause larger effect on the dispersion of the resonance peaks as shown by the wider dispersion of the peaks in both dimensions (Fig. 5F, circle indicated). Interestingly, some peaks disappear in the spectra while some new peaks appear, possibly due to formation of the soluble aggregates. The NMR experiments strongly support that the GAV peptide can induce conformational change and soluble aggregation of G68E mutant. Combined with CD and other experiments, we propose that G68E mutant can form considerable

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amounts of b-sheet structure in the soluble oligomers or even fibrils with the assistance of the GAV peptide. 3.5. The GAV peptide fails to trigger aggregation of the GAVdeficient mutant of a-Syn Since the GAV-deletion mutant (D66–74) loses the capacity of fibrillization, we examined whether the GAV peptide could restore the aggregation behavior in this a-Syn mutant. Unfortunately, there is no observable turbidity in the mixture of D66–74 and GAV even after an incubation of over 6 days with continuous shaking at 37 C (Fig. 1B). Both ThT fluorescence assay (Fig. 2B) and SDS–PAGE analysis (Fig. 3D) support the notion that D66–74 does not aggregate into filaments no matter the GAV peptide is present or not. This indicates that the D66–74 mutant has lost not only the capacity of self-fibrillization but also the potential of being restored to form fibrils by the GAV peptide. Taken together, we conclude that the GAV motif is crucial for a-Syn aggregation, and the interplay between the GAV peptide and the core regions in a-Syn may accelerate the aggregation process and stabilize the fibrils.

Fig. 4. AFM images of the fibrils from G68R or G68E mutant with the assistance of the GAV peptide. (A) Peptide-free G68R sample. (B) The GAV peptide-assistant fibrillization of G68R. (C) Peptide-free G68E sample. (D) The GAV peptide-assistant fibrillization of G68E. The peptide-free samples were incubated for 15 days and the samples with the GAV peptide were incubated for only 6 days. The numerical values beside the graphs are the average heights of the fibrils. Scale bar, 200 nm.

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Fig. 5. 1H–15N HSQC spectra of G68E mutant showing the conformational changes during the GAV peptide-induced aggregation. (A) HSQC spectrum of fresh G68E. (B) HSQC spectrum of G68E after incubation of 7 days. (C) HSQC spectrum of G68E recorded immediately after addition of the peptide in a 1:2 molar ratio. (D) HSQC spectra of sample (C) recorded after incubation of 7 days. (E) As in (C), except for the molar ratio of 1:10. (F) As in (E), except for incubation of 7 days. For NMR experiments, 200 lM (3.0 mg/mL) of 15N-labeled G68E was incubated with 0.4 mM (0.32 mg/mL) or 2.0 mM (1.6 mg/mL) peptide in 100 mM phosphate buffer (pH 6.5) at 37 C for 7 days. The peaks with a circle (in D and F) indicate large chemical-shift changes as compared with those of the peptide-free (A and B) or non-incubation (C and E) samples. All the samples in the presence of the peptide remained clear after acquisition of the HSQC spectra.

H.-N. Du et al. / FEBS Letters 580 (2006) 3657–3664

4. Discussion Aggregation or amyloid formation is generally a result of conformational alternation (such as exposure of hydrophobic residues) of a natively folded protein or peptide when it is subjected to abnormal conditions such as a mutation or truncation [1,9]. Even a native peptide chain, such as a-Syn and PrP, is occasionally found to transform into amyloid fibrils under normal conditions [15]. The GAV motif, which is mainly comprised of flexible Gly and relatively hydrophobic residues (Val, Ala), is critical for fibrillization of a-Syn and possibly for that of Ab and PrP [7,8,21,27]. This particular motif is exactly located in the ‘‘aggregation-prone’’ regions in several amyloidogenic proteins as predicted by aggregation propensity profiles [28]. Charged mutation (such as G68E and G68R) impedes fibril formation of a-Syn, but the mutants are still able to form oligomers or protofibrils [21]. The present data show that fibrillization of G68R is more easily induced by the GAV peptide than that of G68E mutant. It is probably because that G68R has more intrinsic ability of oligomerization than G68E in the peptide-free manner (Fig. 4A and C). Our previous study showed that one group of a-Syn mutants (V66R and G68R) can generate small short filaments with different sizes while another group (G68E, V70R/V71R) only forms small oligomers [21]. We propose that the GAV peptide interacts with the GAV core regions of a-Syn or its mutants by forming hydrophobic patches. This is also evidenced by the NMR experiment that the GAV peptide can trigger the conformational change of G68E mutant. When forming fibrils, the GAV peptide chains are entirely embedded into the existing cross b-sheet core. Formation of the mingled b-strands may facilitate elongation of the filaments. Because D66–74 and GAR peptide are deficient of the critical GAV motif, they may have lost the ability of b-sheet formation by intermolecular chain–chain interactions between protein and peptide. Therefore, fibrillization of D66–74 cannot be restored by the GAV peptide or the GAR peptide fails to prompt a-Syn aggregation. The homologous GAV peptides can not only accelerate the fibrillization of WT a-Syn but also more interestingly trigger the fibrillization of charge-incorporated mutants. While the manuscript was in preparation, El-Agnaf et al. [29] reported identification of residues 64–100 of a-Syn as the binding region responsible for its self-aggregation. They designed some a-Syn inhibitors (ASI) based on residues 69–72 of a-Syn with additional arginine residues in both termini (such as RGAVVTGR-NH2), and found that these inhibitory peptides interact with full-length a-Syn and block its self-assembly. This agrees with our previous work that the hydrophobic region 66– 74 (namely GAV motif) is critical for its fibrillization and cytotoxicity [21], and the present result that the interplay between the homologous GAV peptide and the core regions of a-Syn may accelerate the aggregation process and stabilize the fibrils. This is also supported by the fact that the a-Syn aggregation is indeed accelerated by the homologous GAV peptides, and this process is dependent on the peptide sequence. On the other hand, it seems increasingly likely that the early oligomers or protofibrils during fibrillization process are actually the major cytotoxic species responsible for neural cell death, whereas the mature fibrils, such as Lewy bodies, are the inert metabolic products that may protect cells against impairment [30]. This implies that reduction of the oligomeric intermediate by inhib-

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iting protein oligomerization [31–33] or accelerating fibrillization via modified homologous peptides based on the native sequences of the amyloidogenic proteins might be a practicable strategy for therapeutic usage in neurodegenerative diseases. Consistent with this idea, the cell-permeable inhibitor using the polyarginine peptide delivery system has been developed for suppressing the cellular impact of a-Syn aggregation [29]. Therefore, our finding that the homologous peptides can interact with a-Syn and accelerate its fibrillization could be potentially utilized in the development of peptidomimetic drugs that convert the toxic oligomers into inert mature fibrils in vivo to treat the related neurodegenerative diseases. Acknowledgments: We are grateful to Drs. Da-Fu Cui, Lin Tang and Jian-Hua He for the technical assistance and valuable discussion. This work was supported by grants from the National Natural Science Foundation of China (NSFC30570377), the 863 Hi-Tech Program (2002BA711A13), the Chinese Academy of Sciences (KSCX1-SW-17, KSCX2-SW-209) and the Shanghai Commission of Science and Technology (03JC14081).

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