Inhibition of a-synuclein fibrillization by dopamine analogs via reaction with the amino groups of a-synuclein Implication for dopaminergic neurodegeneration Hong-Tao Li 1 , Dong-Hai Lin 2 , Xiao-Ying Luo 1 , Feng Zhang 3 , Li-Na Ji 1,3 , Hai-Ning Du 1 , Guo-Qiang Song 2 , Jun Hu 3,4 , Jia-Wei Zhou 1 and Hong-Yu Hu 1 1 Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 2 Shanghai Institute of Materia Medica, Shanghai, China 3 Shanghai Institute of Nuclear Research, Shanghai, China 4 Bio-X Research Center, Shanghai Jiaotong University, Shanghai, China Parkinson’s disease (PD) is a common movement dis- order characterized by degeneration of dopaminergic neurons and deposition of fibrillar Lewy bodies com- prising primarily a-synuclein (a-Syn) in the substantia nigra [1–4]. A growing body of evidence strongly supports the theory that formation of a-Syn fibrils and dopamine (DA) metabolism are closely associ- ated with the pathogenesis of this fatal disease [5–7]. These findings imply an intrinsic link between the presynaptic a-Syn protein and the DA molecule [8], a synaptic neurotransmitter that functions in signal transmission. Recent research has focused on oxida- tive stress in brain, DA metabolism and dysfunction of a-Syn in synapses, in an attempt to elucidate an overview linking these important biological processes. Conway et al. [9] reported that DA stabilized the a-Syn protofibrils by forming a DA-a-Syn adduct. They proposed that DA could react with the phenol Keywords a-synuclein; dopamine; inhibition; fibrillization; Parkinson’s disease Correspondence H Y. Hu, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, P. R. China Fax: +86 021 54921011 Tel: +86 021 54921121 E-mail: hyhu@sibs.ac.cn (Received 28 March 2005, revised 20 May 2005, accepted 25 May 2005) doi:10.1111/j.1742-4658.2005.04792.x Fibrillization of a-synuclein (a-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkin- son’s disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit a-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of a-Syn chain, generating a-Syn–quinone adducts. It is likely that the amino groups of a-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked a-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact a-Syn, suggest- ing that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of a-Syn with DA is relevant to the selective dopaminergic loss in PD. Abbreviations Ab, amyloid b-protein; AFM, atomic force microscopy; CA, catechol; DA, dopamine; DAQ, dopamine-quinone; EGCG, (–)-epigallocatechin gallate; GAV, a peptide motif with a homologous sequence of VGGAVVAGV; HQ, hydroquinone; HSQC, heteronuclear single quantum coherence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, nonamyloid component; NBT, nitroblue tetrazolium chloride; pNP, p-nitrophenol; PrP, prion protein; pXG, p-xylylene glycol; Q, p-benzoquinone; a-Syn, human a-synuclein; ThT, thioflavin T. FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS 3661 groups of tyrosine residues in a-Syn resulting in dityrosine linkages [10] and stabilize a-Syn oligomers or protofibrils, which are more toxic than the mature fibrils to neural cells. Although the biological function of a-Syn has not been fully understood, it is characterized by fibrillar assembling both in vivo and in vitro [2,11,12]. The protein contains an apolipoprotein-like amphipathic N-terminus with 5–7 repeats of KTKEGV sequence, an acidic C-terminus and the so-called nonamyloid component (NAC) region [13]. The N- and C-terminal regions are not directly responsible for a-Syn fibrilliza- tion, whereas the central hydrophobic region is likely to be the nucleation core [14,15]. We have identified a novel GAV motif that is essential to fibrillization of a-Syn as well as other amyloidogenic proteins [16] and found that the natively unfolded structure of the pro- tein also plays an important role in the fibrillization process (unpublished data). Study of inhibition of fibrillization may provide insight into understanding of the a-Syn fibrillization (Lewy-body formation) and the related cytotoxicity (dopaminergic loss). In addition, research on the fibrillization inhibitors may help us elucidate the pathogenesis of DA-related neurodegen- erative diseases and discover potential probes and drugs for clinical diagnosis and treatment. We tested DA and some polyphenol analogs and found that they inhibit a-Syn fibrillization with great efficacy in vitro. These polyphenols can react with and covalently modify the a-Syn molecule through linking to the amino groups of the protein. Cross-linked dimers or oligomers by DA or other analogs were also identified and found to be toxic to PC12 cells. Results Inhibition of a-Syn fibrillization by dopamine analogs In a manner similar to other amyloidogenic proteins, a-Syn readily aggregates into fibrils in vitro. Under the conditions of incubation at 37 °C, it normally takes six days to grow into the regular mature fibrils [16]. The time courses of the fibrillization processes of a-Syn incubated with the DA-analog compounds were monitored by a thioflavin (ThT) fluorescence assay [17]. Figure 1 displays inhibition of a-Syn fibrillization by catechol-like polyphenols. Hydroquinone (HQ) and catechol (CA), two diphenol isoforms, significantly inhibit the fibrillization of a-Syn (Fig. 1B). They can completely destroy a-Syn fibrillization at a concentra- tion as low as 50 lm, compared with the protein con- centration of 200 lm. DA and l-dopa, two natural structural analogs of catechol, also inhibit a-Syn fibril- lization with an equimolar amount (Fig. 1C) as repor- ted previously [9]. p-Nitrophenol (pNP) and ascorbic acid (vitamin C) are also efficacious inhibitors for the processes (Fig. 1D). The electron attraction property of the nitro group in pNP and the conjugated enol form of vitamin C make them a bit acidic and reactive to oxidation. Chemically, they are similar to diphenol compounds. Interestingly, p-benzoquinone (Q), the oxi- dation product of HQ, can equally suppress the fibrilli- zation process. We also examined polyphenols isolated from green tea. The polyphenol mixture also effectively inhibits a-Syn fibrillization (data not shown), and (–)- epigallocatechin gallate (EGCG), a major component of green tea polyphenols, gives an IC 50 of  20 lm . Moreover, p-xylylene glycol (pXG) was used as a neg- ative control. It has no inhibiting effect on a-Syn fibril- lization even at millimolar amounts (Fig. 1E). These data demonstrate that the inhibition results from spon- taneous oxidation products of these polyphenols, rather than from binding of the aromatic rings. Dopamine-quinone (DAQ) has been reported to react with a-Syn leading to inhibition of a-Syn fibril- lization but prolonging the lifetime of the protofibril- lar intermediates [9]. As revealed by AFM [18], a-Syn aggregates into abundant regular filaments after incu- bation for six days. However, in the presence of inhibitors such as HQ and DA during the incubation process, a-Syn fails to form regular fibrils. Only small oligomers are visualized dispersing in the images (Fig. 1F). This observation demonstrates that DA analogs inhibit formation of a-Syn fibrils but stabilize the oligomers or protofibrils in the intermediate states. Reactions of a-Syn with quinones Polyphenols can be spontaneously oxidized into qui- nones by dissolved oxygen in the buffer at atmo- sphere. Reactions of these quinones with a-Syn result in the increases of absorbance at 280 nm and 345 nm in the UV spectra (see below). Under inhibitory con- ditions, the oxidation products of polyphenols (CA, HQ and Q) reacting with a-Syn show the increase of UV absorption in the position of residual monomeric forms in size-exclusion chromatography (SEC) profile (Fig. 2A), suggesting that these fractions are gener- ated by cross-linking of a-Syn with the quinones [9]. From the UV absorption of SEC profile, it is suggest- ive that the reaction of Q or HQ with a-Syn gives rise to high amount of monomeric adducts. Besides the monomers, the reaction products also contain fractions with larger molecular masses, mostly in Reaction of a-synuclein with dopamine analogs H T. Li et al. 3662 FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS dimeric and trimeric forms. Intriguingly, DA quinone reacts with a-Syn to generate large molecular mass fractions, mostly oligomers or protofibrils. SDS ⁄ PAGE analysis of the fractions from the SEC profiles reveals a molecular ladder of oligomeric forms (Fig. 2B). The reaction product of DAQ contains large amount of oligomers, while those of other qui- nones are mainly dimers and slightly trimers. These oligomers are cross-linked species as they are resistant to dissociation by SDS and 8 m urea (Fig. 2C). MS analysis of the oligomeric fraction from the Q reac- tion product also reveals a large amount of a-Syn- adduct dimers (Fig. 2D), in which one protein dimer may attach several quinones to give a molecular mass mixture (the r.m.m. of a-Syn dimer is  28 920 Da). These observations suggest that the quinones react with a-Syn to generate quinone adducts and cross- linked dimers or oligomers. Quinones are cross-linking to a-Syn To ascertain that these DA analogs are cross-linking to a-Syn, we determined the molecular masses of the monomeric forms from the reaction products by ESI- MS (Fig. 3). The monomeric fraction of the reaction products shows a bundle of peaks with different molecular masses, while the intact a-Syn gives a major peak with a molecular mass of 14 458 Da. Table 1 dis- plays the molecular masses and the proposed pattern of the monomeric adducts by these reactions. Reaction of a-Syn with these DA analogs gives a group of adducts with molecular masses larger than intact a-Syn (expectedly 14 460 Da). For the Q reaction, the additional mass is an exact multiple of 104 (104 n) Daltons, which is in agreement with the molecular mass of the Q individual after reaction with two active groups (108–2 · 2 Da). The patterns for the incremen- 400 nm 400 nm 400 nm A BC D F E Fig. 1. Inhibition of a-Syn fibrillization by polyphenols. (A) Structures of the com- pounds. HQ, hydroquinone; CA, catechol; Q, p-benzoquinone; pXG, p-xylylene glycol; pNP, p-nitrophenol; DA, dopamine; VC, vita- min C. (B) HQ and CA in different concen- trations. (C) DA and L-dopa. (D) Q, VC and pNP. (E) pXG. The mixtures of a-Syn and the compounds were incubated for several days, and then the fibrillization at different intervals was measured by ThT fluorescence assay. The concentration of a-Syn was 200 l M and the fibrillization of a-Syn alone was as a comparison. Data were represen- ted as means ± SEM. (F) Atomic force microscopic images of a-Syn fibrils. a-Syn (200 l M) was incubated with 200 lM of HQ (middle) or DA (right) for 6 days. The a-Syn alone sample was as a control (left). All graphs are topographical height images of 2 lm 2 in area and the scale bar represents 400 nm. H T. Li et al. Reaction of a-synuclein with dopamine analogs FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS 3663 tal masses for the reactions of HQ and CA can also be expressed as 104 n +16 m (where n and m are inte- gers, normally n £ 6, m £ 4). The additional molecular mass of 16 Da implies that the protein has been oxi- dized by reactive oxygen species generated by auto- oxidation of the polyphenols during incubation process. The most likely sites for the oxidation are within the methionine residues, where oxygen atoms easily adduct to the thioether groups to be sulfoxides [19]. a-Syn 1)60 , the nonamyloidogenic variant of a-Syn, can also react with Q, resulting in the products with additional mass of multiple 104 Da. From Fig. 3 and Table 1, we found that the predominant reaction products of Q or HQ are a-Syn-Q adducts, while those of CA are mainly methionine oxidized a-Syn with small amount of a-Syn-CA adducts. This observation is consistent with the UV absorption enhancement in SEC profiles (Fig. 2A). Unexpectedly, we failed to detect the DA adduct in the monomeric form by mass spectrometry. To confirm that reaction of DA B D C A Fig. 2. Cross-linked oligomerization of a-Syn by DA analogs. (A) SEC analysis of the reaction products showing the large molecular mass fractions. The graphs display incubation of a-Syn alone (black), the reactions of a-Syn with DA (red), CA (green), HQ (purple) and Q (blue), respectively. a-Syn (200 l M) was incubated with 1 mM of DA analogs for 24 h, and then the reaction mixtures were analyzed by means of SEC. (B) SDS ⁄ PAGE graph of the fractions from SEC separation of the reaction products of a-Syn with Q (up) or DA (down). The fractions collected from the elution profile at around 19 min and 23 min are dominantly oligomers and dimers, respectively. Samples were loaded on an SDS ⁄ PAGE (15% polyacrylamide gel) with silver staining. Mr, molecular mass marker; M, monomer; D, dimer; T, trimer; O, oligomer. (C) SDS ⁄ PAGE graph of the reaction products from a-Syn (200 l M) with 1 mM of DA analogs showing the covalently modified adducts. The reaction mixtures were dissolved in a loading buffer with (right lane) or without (left lane) 8 M urea. An equal amount of the proteins was loa- ded in each lane of the gel. The a-Syn alone sample incubated for 1 day was also subjected to SDS ⁄ PAGE as a control, which shows no dimer or oligomer in the gel. (D) MALDI-TOF MS spectrum of the large molecular mass fraction from the reaction of a-Syn with p-benzo- quinone. The sample was isolated from the reaction mixture by SEC. The small peaks (M) with molecular masses around 15 kDa are the contaminants of monomeric form in the dimeric fraction. M: monomer, r.m.m. of  14 460 Da; D: dimer, r.m.m. of  28 920 Da. The peaks with larger molecular masses indicate different species of a-Syn–quinone adducts. Reaction of a-synuclein with dopamine analogs H T. Li et al. 3664 FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS with a-Syn produces mostly large molecular mass adducts, we performed SDS⁄ PAGE analysis by redox- cycling staining [20] for detecting quinoproteins (Fig. 4). The quinoproteins of the DA-adduct are largely involved in the oligomeric forms, while those of other products (Q-, HQ- or CA-adduct) are in monomeric and dimeric forms. This is in good agree- ment with the result from Coomassie Blue staining for protein bands. The results suggest that DA attach to a-Syn chain through DAQ, leading to formation of the oligomeric adducts while other analogs form quinoproteins in monomeric or dimeric form. Fig. 3. Electrospray MS analysis of the monomeric a-Syn–quinone adducts. The reaction mixture of a-Syn (200 l M)and 3m M of Q (middle) or 5 mM of HQ (right) was incubated for 24 h and the monomeric fraction was isolated by SEC for MS analy- sis. The MS graph of fresh a-Syn was as a control (left). Table 1. Molecular masses of the monomeric adducts as determined by electrospray mass spectrometry. Molecular masses were of the peaks detected by ESI-MS. Pattern shows incremental molecular masses of the reaction products. Reaction Molecular masses (Da) Pattern a-Syn 1)60 6189.5 ¼ 6188.2 +1.3 6188.2 a-Syn 1)60 + Q, 1 h 6189.5 ¼ 6188.2 +1.3; 6293.3 ¼ 6188.2 +104 +1.1; 6188.2 + 104 n 6396.8 ¼ 6188.2 +208 +0.4; 6499.5 ¼ 6188.2 +312–0.7; a-Syn 14 458 ¼ 14 460–2 14 460 a-Syn + Q, 1 h 14 565.5 ¼ 14 460 +104 +1.5; 14 668.8 ¼ 14 460 +208 +0.8; 14 460 + 104 n 14 764.0 a-Syn+ Q, 24 h 14 563.8 ¼ 14 460 +104–0.2; 14 668.5 ¼ 14 460 +208 +0.5; 14 460 + 104 n 14 772.8 ¼ 14 460 +312 +0.8 a-Syn + HQ, 24 h 14 562 ¼ 14 460 +104–2; 14 580 ¼ 14 460 +104 +16; 14 460 + 16 m + 104 n 14 666 ¼ 14 460 +208–2; 14 682 ¼ 14 460 +208 +16–2 14 787 ¼ 14 460 +312 +16–1; 14 800 ¼ 14460 +312 +32–4; 14 823 ¼ 14 460 +312 +48 +3; 14 909 ¼ 14460 +416 +32 +1; 15 043 ¼ 14 460 +520 +32 +1 a-Syn + CA, 24 h 14 477 ¼ 14 460 +16 +1; 14 490 ¼ 14 460 +32–2; 14 460 + 16 m + 104 n 14 507 ¼ 14 460 +48–1; 14 734 ¼ 14 460 +208 +48 +2; 14 804 ¼ 14 460 +312 +32; 14 993 ¼ 14 460 +520 +16–3; 15 119 ¼ 14460 +624 +32 +3 a-Syn + DA, 96 h 14 491 ¼ 14 460 +32–1 a 14 460+16 m a No DA-adduct was detected in the monomeric form. H T. Li et al. Reaction of a-synuclein with dopamine analogs FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS 3665 Quinone linkage through amino groups of a-Syn The possible reactive sites for reaction with Q in a pro- tein chain are the side chains of Cys, Tyr, Lys and Met residues [21]. a-Syn contains no Cys residue; the Tyr residues are presumably reported to be the most potent candidates for the reaction [9]. The molecular masses of the reaction adducts with the increase of multiple 16 suggest that Met residues are the oxidation sites for oxygen. We compared the aromatic region (6.0–8.0 p.p.m.) of 1 H- 1 H COSY spectra of the intact and quinone-modified a-Syn (data not shown). The result shows that the phenol groups of Tyr residues are unlikely to be the Q linkage sites, because no chemical-shift disturbance occurs in the aromatic region. Mutation of tyrosine to phenylalanine also sug- gested that the tyrosine residues are not required for protein cross-linking [22]. Quinones readily react with a-Syn generating two absorption peaks at 280 nm and 345 nm in the UV spectra (Fig. 5A). Our experiments show that Q can also react with a-amino group of Gly, Tyr or Lys, but not with N-acetylglycine, producing a large absorption peak at 345 nm (Fig. 5B). Instead, reaction of a-Syn with DA generates a broad shoulder around 345 nm in the UV spectra (Figs 5C,D). It is likely that DAQ reacts with the amino group of DA molecule resulting in formation of melanin that also contributes to the spectra [23]. These results suggest that the quinone molecule reacts with a-Syn by attaching to the e-amino groups of Lys residues or the a-amino group in the protein N-terminus. To confirm this, we performed 1 H- 15 N HSQC spectra in an 15 N-labeled a-Syn sample. The chemical shifts of amides in intact a-Syn exhibit small dispersion in the 1 H dimension, suggesting that a-Syn is natively unstructured. No peak appeared in the HSQC spectrum for the e-amino groups of Lys residues due to the fast exchange with solvent. When a-Syn reacts with Q (Fig. 6A) or DA (Fig. 6B), three additional peaks emerge in the 7.7–8.0 p.p.m. region ( 1 H dimension) as compared with that of intact a-Syn. This suggests that at least three amino groups of an a-Syn molecule are modified by Q or DA to generate imino groups that link to the aromatic ring of quinone and the side chains of the protein. The NMR experi- ments provide convincing evidence in support of the reaction between amino groups of Lys residues and Q or DAQ (the oxidation product of DA). As there are 16 amino groups (15 Lys, one a-amino group) in an a-Syn molecule, it is worthy of identifying the three to four reactive Lys residues in a-Syn. DA linkage retains the cytotoxicity of a-Syn The previous studies have shown that a-Syn fibrilliza- tion is toxic to neural cells [5,16]. We examined the effect of DA-modified a-Syn on cultured PC12 cells by MTT assay [24]. Figure 7 shows the percentage of MTT reduction in cell cultures vs. the incubation time. The incubated a-Syn sample has a significant impact on the cell viability. It seems that 2-day incubated a-Syn is more toxic to the cells than the fresh sample or the samples after incubation for 4–6 days. This sug- gests that prolonging of incubation may decrease the amount of oligomeric intermediates and prompt for- mation of the mature fibrils that may have less toxi- city. DA alone has a small effect on the cell viability as reported previously [25]. In a manner similar to A B Fig. 4. Redox-cycling staining of quinone-modified a-Syn. a-Syn (200 lM) was incubated alone (control) or with 1 mM of DA, CA, HQ or Q at 37 °C for 24 h then subjected to SDS ⁄ PAGE (15% polyacrylamide gel). (A) Redox-cycling staining for quinoproteins. (B) Coomassie Blue staining for proteins. The a-Syn protein (control) cannot be stained with quinone-specific reagent while it can with Coomassie Blue staining. Mr, molecular mass marker; M, monomer; D, dimer; T, trimer; O, oligomer. Reaction of a-synuclein with dopamine analogs H T. Li et al. 3666 FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS intact a-Syn, the DA-modified sample also presents significant effect on the cell viability. Similarly, after removal of excess DA, the DA-modified protein (mixture of monomer and oligomer), also retains the cytotoxicity, supporting the hypothesis that the cyto- toxicity primarily arises from the reaction adduct or the a-Syn oligomers rather than from free DA or the a-Syn fibrils. Although DA modification inhibits a-Syn fibrillization, the MTT reduction experiment suggests that DA-modified a-Syn has significant cytotoxicity on PC12 cells. We also isolated the monomeric form of DA-modified a-Syn by SEC and examined its cyto- toxicity. The result shows that the monomeric form of DA-modified a-Syn product exerts a slightly toxic effect on PC12 cells (Fig. 7B). This implies that the oligomeric fraction may be the major element that cau- ses the cytotoxicity. Reaction with DA inhibits a-Syn fibrillization but stabilizes the oligomers, which retains the cytotoxicity. Discussion Many compounds that inhibit protein amyloidogenesis are the derivatives of CA or HQ, such as DA [9,26] and flavonoid [27] for a-Syn, apomorphine [28] and rif- ampicin [29] for amyloid b protein, and anthracycline analogs for prion protein [30] and other amyloid fibrils [31]. DA and other polyphenols are natural com- pounds that are readily oxidized to quinone forms when exposed to an oxygen atmosphere. Interestingly, vitamin C (a biological antioxidant) and EGCG (a green tea polyphenol of antioxidant role) are also effective inhibitors for the a-Syn fibrillization. Unlike DA, CA and Q (or HQ) can inhibit a-Syn fibrillization Fig. 5. Spectrophotometric characterization of the reactions of DA analogs with a-Syn and some amino acids. (A) UV-vis spectra of a-Syn with DA, CA, HQ and Q after reaction for 24 h. The a-Syn alone sample was as a control. The concentration of a-Syn was 200 l M and that of the compounds was 1 m M. (B) Time courses of the reactions of Q with a-Syn (circle) and some amino acids as recorded at 345 nm. The amino acids are Gly (m), Lys ( ), Tyr (.) and N-acetylglycine (r), respectively. The Q-alone sample (w) was used as a control. The concen- tration was 4 m M for Q, 200 lM for a-Syn and 2 mM for different amino acids. (C) Absorption spectra of a-Syn (200 lM) reacting with DA (2 m M). The absorbance at 345 nm increases with the incubation time (0–24 h). (D) Time course of the reaction of a-Syn (200 lM) with DA (2 m M)(d) as recorded at 345 nm. The DA alone sample ( ) was used as a control. H T. Li et al. Reaction of a-synuclein with dopamine analogs FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS 3667 with great efficacy, generating mostly monomers or dimers but not large molecular mass oligomers. Based on the novel reaction and the inhibitory feature of quinone moiety, chemically synthetic small compounds may be applicable to the development of potential probes for monitoring the fibrillization processes of Fig. 7. Cytotoxicity of DA-modified a-Syn. (A) Time course of the MTT reduction of different a-Syn forms. The mixture of a-Syn and DA in different incubation time was performed on cytotoxicity assay on PC12 cells (Syn + DA). To exclude the possibility that DA affects the assay, excess DA was removed from the protein mixture by desalting column (del DA). Normally, equal molar of DA (300 l M) was used for the incubation. The percentage of MTT reduction represents the cell viability after treatment with incubated protein aggregate. The MTT reduction ability of the cell culture after addition of a-Syn or DA was also measured as a comparison. The concentration of all a-Syn forms for the MTT assay was 10 l M. Data represented are mean ± SEM, n ¼ 3. Statistically significant differences (*P<0.05; **P<0.01) are indicated for comparisons of aged against fresh sample by t-test. (B) Cytotoxicities of different a-Syn forms. After reaction for 24 h, the monomeric fraction was isolated by SEC and the concentration was determined by amino-acid analysis. The cell toxicity of the reaction pro- duct after removal of excess DA (a mixture of monomer and oligomer) was also measured for a comparison (del DA). A 115 120 125 130 8.5 8.0 7.5 1 H ( pp m) 15 N (ppm) 1 H ( pp m) 7.0 8.5 8.0 7.5 7.0 B Fig. 6. 1 H- 15 N HSQC spectra of the reactions of a-Syn with quinone and DA. (A) Overlay of the 1 H- 15 N HSQC spectra of a-Syn (black) and the reaction product of a-Syn and Q (red). (B) Overlay of the 1 H- 15 N HSQC spectra of a-Syn (black) and the reaction product of a-Syn and DA (red). For NMR experiments, 300 l M of 15 N-labeled a-Syn was incubated with 3 mM quinone or DA in 10 mM NaCl ⁄ P i (pH 6.5) at 37 °C for 24 h. There is no significant change of the peaks in the amide region except for three extra peaks (arrows indicated) emerging in each spectrum. The peaks appearing in the amide region of HSQC spectra reflect formation of imino groups during reaction of the side-chain amino groups with Q or DAQ. Reaction of a-synuclein with dopamine analogs H T. Li et al. 3668 FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS a-Syn in vivo and antiamyloid drugs for clinical treat- ment for PD. Recently, Zhu et al. [27] have provided an assump- tion that flavonoid baicalein inhibits a -Syn fibrilliza- tion by reaction of baicalein quinone with a lysine side-chain of a-Syn to form a Schiff base, and the additional mass of the product should be 90 Da. Our MS results show that the a-Syn–quinone adducts from CA or Q (or HQ) possess an additional mass of muti- ple 104 Da (Table 1). It is likely that the amino groups of a protein undergo nulceophilic attack on the highly reactive oxidation products (commonly quinones) to form imino bonds (R 1 -NH-R 2 ) (Figs 5 and 6), being the additional mass of exact 104 Da. According to Fis- cher and Schrader reported in 1910 and many other literatures [32,33], the general reaction for quinone- adduct formation can be schematically represented in Fig. 8. One or two lysine residues react with a quinone molecule, and the reaction product is quinone-Lys adduct normally with a cross-linking. If the lysine resi- dues are from different a-Syn molecules, intermole- cular cross-linking is generated within the covalently adducted oligomers. The DA-a-Syn reaction products are primarily oligomers with large molecular masses rather than monomers or dimers, probably because DAQ reacts with the amino group of DA molecule to form melanin [23] that further covalently cross-links DA-a-Syn adducts to form more complicated DA- a-Syn oligomers. It is known that protein fibrillization causes the dys- function of cell impairment. It is also proposed that the oligomeric fractions during fibrillization process, but not the mature fibrils, are the major cytotoxic spe- cies that cause PD [34,35]. a-Syn contains 15 Lys, four Met and four Tyr residues but no Cys residue in its amino acid sequence. A recent report shows that cys- teine substitutions at 39 and 125 positions in a-Syn increase protein aggregation and cellular toxicity [36]. The high frequency of Lys residues in the N-terminus and several repeats of KTKEGV sequence provide a high probability for the reaction with DAQ through the side-chain amino groups of Lys residues. More- over, the natively unfolded structure of a-Syn [37] makes these reactive side chains exposed to solvent, which accelerates the reaction with quinones in vitro and probably in vivo. DA is synthesized in the cytoplasm of dopaminergic neurons and stored in the presynaptic vesicles [4]. The re-uptake of DA to presynapse is undertaken by a membrane protein, dopamine transporter (DAT), which is in turn regulated by cytosolic a-Syn [38,39]. This is a possible way that a-Syn regulates DA meta- bolism in the normal presynaptic processes [40,41]. Our finding provides another possibility that a-Syn interferes with DA metabolism by forming DAQ adducts through its amino groups. a-Syn is highly expressed in the presynaptic terminals, and the DA vesicles are also accumulated in the cytosol under Fig. 8. Schematic representation for the reaction of lysine side chain with quinones. The quinones can be regarded as the spontaneous oxi- dation product of polyphenol analogs, and the amino groups are from side chains of lysine residues in proteins and other biological amines. 1, HQ; 2, Q; 3 & 4, adducts of one or two amino groups with HQ or Q; 5, CA and its derivatives; 6 & 7, adducts of one or two amino groups with CA and its derivatives; The molecular mass of 2 is 108 Da, and the additional masses of 3 and 4 are 106 and 104 Da, respectively. H T. Li et al. Reaction of a-synuclein with dopamine analogs FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS 3669 normal conditions. There is a stress of highly reactive oxygen species in human brain, where the catechol moiety of free cytosolic DA is inevitably oxidized to the quinone form [42]. High concentration of a-Syn not only prompts self-assembly of the protein that is destined to accumulation in oligomeric states (proto- fibrils) or in Lewy bodies, but also provides an oppor- tunity for interaction with DA vesicles [43]. Thus, the a-Syn oligomers permeabilize the DA vesicles, thereby leading to leakage of DA molecules from the vesicles [8,44]. This greatly improves the probability that the two molecules meet and react in the presynaptic cyto- sol. In turn, reaction with DA makes a-Syn covalently cross-linked and stay in an oligomeric state. The pore- like oligomeric intermediates (protofibrils) of a-Syn- DA adducts further strongly attacks on and disrupt membranes [45]. Thus, the reaction cascade of a-Syn and DA on synaptic vesicles or the cell membrane might damage cellular function and cause selective dopaminergic neurodegeneration. Experimental procedures Materials Thioflavin T (ThT), catechol (CA), hydroquinone (HQ), dopamine (DA) and l-dopa were purchased from Aldrich. p-Xylylene glycol (pXG, C 6 H 4 (CH 2 OH) 2 ) was a product of Tokyo Chemical Industry (Tokyo, Japan). (–)-Epigallocate- chin gallate (EGCG) was from Sigma (St Louis, MO, USA), and N-acetylglycine was from Fluka (Buchs, Switzerland). [ 15 N]Ammonium chloride was obtained from Cambridge Isotope Laboratories and Nitroblue tetrazolium chloride (NBT) was from Bio Basic Inc (Markham Ontario, Canada). All other reagents were of analytical quality. Expression and purification of a-Syn and a-Syn 1)60 a-Syn and a-Syn 1)60 genes were subcloned into the pET-3a vector, and the respective proteins were expressed in Escherichia coli BL21 (DE3). 15 N-labeled a-Syn was expres- sed in M9 minimal media supplemented with [ 15 N]ammo- nium chloride. Labeled or unlabeled human recombinant a-Syn was prepared as described previously [16]. The trun- cated protein a-Syn 1)60 was purified through a CM-sepha- dex C25 cation-exchange column and a FPLC superose-12 column (Pharmacia, Uppsala, Sweden). Inhibition of fibrillization processes The time course of a-Syn fibrillization was measured by ThT fluorescence assay, in which the fluorescence intensity enhancement reflects the degree of fibrillization [17]. All chemical compounds were dissolved in NaCl ⁄ P i buffer (100 mm phosphate, 100 mm NaCl, pH 7.0) as stock solu- tions. The concentration of a-Syn was adjusted to 200 lm and then sterilely filtered through 0.22 lm filters to remove any granular matter. In the presence of different concentra- tions of inhibitors, the samples were incubated in 1.5 mL sterile tubes with continuous shaking at 37 °C. The ThT fluorescence assay at various time points was performed on a Hitachi F-4010 fluorophotometer. Atomic force microscopy The morphologies of a-Syn samples after incubation for 6 days with or without DA analogs were visualized by AFM imaging as described previously [16,18]. SEC and MS analysis Either a-Syn or a -Syn 1)60 (200 lm) was incubated with the polyphenols (Q, HQ, CA or DA) at 37 °C with continuous shaking for 1–24 h, followed by centrifugation at 16 000 g for 5 min, and then aliquots of the reaction mixture were loaded onto a superpose-12 HR30 ⁄ 10 column (Pharmacia). The molecular masses of the fractions were determined by electrospray (Finnigan-LCQ-Classic, San Jose, CA, USA) or MALDI-TOF (Bruker, Bremen, Germany) mass spectro- metry (MS). The molecular mass of a-Syn or a-Syn 1)60 was also determined as a control. Redox-cycling staining a-Syn (200 lm ) was incubated alone or with DA, CA, HQ or Q (1 mm)at37°C for 24 h then subjected to SDS ⁄ PAGE on 15% polyacrylamide gel. Proteins were transferred from the polyacrylamide gel onto nitrocellulose membranes at 100 V, 4 °C for 3 h in a buffer of 25 mm Tris ⁄ HCl, pH 8.3, containing 192 mm glycine and 20% (v ⁄ v) methanol. Quinoproteins were detected by staining blots with NBT (0.24 mm in 2 m potassium glyci- nate, pH 10.0) in dark for 1 h as described in the literature [20] with some modification. The blue-purple-stained quino- proteins were photographed. The blots were washed with water and restained for total protein with Coomassie Blue R-250. NMR characterization All NMR experiments were performed on a Varian Unity Inova 600 MHz spectrometer (Varian Inc, Palo Alto, CA, USA). To test potential cross-linking of a-Syn through tyrosine side chains, unlabeled a-Syn (400 lm) in a NaCl ⁄ P i buffer (100 mm phosphate, 100 mm NaCl, pH 7.0, in D 2 O) was incubated with quinone (4 mm)at37°C for 2 days then subjected to a 1 H- 1 H COSY experiment. 15 N-labeled a-Syn (300 lm) in a buffer (10 mm phosphate, 10 mm NaCl, pH 6.5) with 8% D 2 O was incubated with quinone or Reaction of a-synuclein with dopamine analogs H T. Li et al. 3670 FEBS Journal 272 (2005) 3661–3672 ª 2005 FEBS [...]... 3661–3672 ª 2005 FEBS Reaction of a-synuclein with dopamine analogs 9 Conway KA, Rochet JC, Bieganski RM & Lansbury PT (2001) Kinetic stabilization of the a-synuclein protofibril by a dopamine -a-synuclein adduct Science 294, 1346–1349 10 Souza JM, Giasson BI, Chen Q, Lee VM & Ischiropoulos H (2000) Dityrosine cross-linking promotes formation of stable a-synuclein polymers Implication of nitrative and oxidative... 20313–20316 3671 Reaction of a-synuclein with dopamine analogs 22 Norris EH, Giasson BI, Ischiropoulos H & Lee VMY (2003) Effects of oxidative and nitrative challenges on a-synuclein fibrillogenesis involve distinct mechanisms of protein modifications J Biol Chem 278, 27230–27240 23 Asanuma M, Miyazaki I & Ogawa N (2003) Dopamineor l-DOPA-induced neurotoxicity: the role of dopamine quinone formation and... al dopamine (3 mm) at 37 °C for 24 h The Q- or DA-modified a-Syn was then subjected to carry out 1H-15N HSQC experimetns The a-Syn alone samples were set as controls Cytotoxicity assay The cytotoxicities of DA-modified a-Syn aggregates were determined in PC12 cells by MTT reduction assay as described previously [16,24] The percentage of MTT reduction represents the cell viability after treatment with. .. Pathogenesis of Parkinson’s disease: dopamine, vesicles and a-synuclein Nat Rev Neurosci 3, 932–942 5 Xu J, Kao SY, Lee FJS, Song WH, Jin LW & Yankner BA (2002) Dopamine- dependent neurotoxicity of a-synuclein: a mechanism for selective neurodegeneration in Parkinson disease Nat Med 8, 600–606 6 Perez RG, Waymire JC, Lin E, Liu JJ, Guo F & Zigmond MJ (2002) A role for a-synuclein in the regulation of dopamine. .. coupling of a-synuclein to the dopamine transporters accelerate dopamine- induced apoptosis FASEB J 15, 916–926 Wersinger C & Sidhu A (2003) Attenuation of dopamine transporter activity by alpha-synuclein Neurosci Lett 340, 189–192 Wersinger C, Prou D, Vernier P, Niznik HB & Sidhu A (2003) Mutations in the lipid-binding domain of a-synuclein confer overlapping, yet distinct, functional properties in the. .. properties in the regulation of dopamine transporter activity Mol Cell Neurosci 24, 91–105 Wersinger C, Prou D, Vernier P & Sidhu A (2003) Modulation of dopamine transporter function by a-synuclein is altered by impairment of cell adhesion and by induction of oxidative stress FASEB J 17, 2151–2153 Klegeris A, Korkina LG & Greenfield SA (1995) Autoxidation of dopamine: a comparison of luminescent and spectrophotometric... (2003) Role of a-synuclein carboxy-terminus on fibril formation in vitro Biochemistry 42, 8530–8540 16 Du HN, Tang L, Luo XY, Li HT, Hu J, Zhou JW & Hu HY (2003) A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human a-synuclein Biochemistry 42, 8870–8878 17 LeVine H (1999) Quantification of b-sheet amyloid fibril structures with thioflavin... D, Aggarwal A, Teichberg S & Callaway DJ (2002) New class of inhibitors of amyloid-b fibril formation Implications for the mechanism of pathogenesis in Alzheimer’s disease J Biol Chem 277, 42881–42890 29 Tomiyama T, Shoji A, Kataoka K, Suwa Y, Asano S, Kaneko H & Endo N (1996) Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin Its possible function as a hydroxyl radical scavenger... 32 Morrison M, Steele W & Danner DJ (1969) The reaction of benzoquinone with amines and proteins Arch Biochem Biophys 134, 515–523 33 Mason HS & Peterson EW (1955) The reaction of quinones with protamine and nucleoprotamine: N-terminal proline J Biol Chem 212, 485–493 34 Goldberg MS & Lansbury PT (2000) Is there a causeand-effect relationship between alpha-synuclein fibrilli- 3672 H.-T Li et al 35 36... viability after treatment with incubated samples The a-Syn or DA alone sample was served as a comparison Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (NSFC39990600 and NSFC30070165), the Chinese Academy of Sciences (KSCX2-SW-209), the Hundred Talents Project of CAS (D Lin) and Shanghai Commission of Science and Technology (03JC14081) Drs Ai-Xin . Inhibition of a-synuclein fibrillization by dopamine analogs via reaction with the amino groups of a-synuclein Implication for dopaminergic neurodegeneration Hong-Tao. mass of 14 458 Da. Table 1 dis- plays the molecular masses and the proposed pattern of the monomeric adducts by these reactions. Reaction of a-Syn with these