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 th
Trang 11 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.
Trang 2groups 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 37C, 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 IC50 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 gsuggest-ives rise to high amount of monomeric adducts Besides the monomers, the reaction products also contain fractions with larger molecular masses, mostly in
Trang 3dimeric 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
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 l M 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.
Trang 4tal 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-Syn1)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 m M 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 m M 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.
Trang 5with 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
3 m M of Q (middle) or 5 m M 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-Syn1)60 6189.5 ¼ 6188.2 +1.3 6188.2
a-Syn1)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 + 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.
Trang 6Quinone 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 1H-1H 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
1H-15N HSQC spectra in an15N-labeled a-Syn sample
The chemical shifts of amides in intact a-Syn exhibit
small dispersion in the 1H 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 (1H 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
Fig 4 Redox-cycling staining of quinone-modified a-Syn a-Syn (200 l M ) was incubated alone (control) or with 1 m M 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.
Trang 7intact 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 l M for a-Syn and 2 m M for different amino acids (C) Absorption spectra of a-Syn (200 l M ) 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 l M ) with DA (2 m M ) (d) as recorded at 345 nm The DA alone sample ( ) was used as a control.
Trang 8with 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
15N (ppm)
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 of15N-labeled a-Syn was incubated with 3 m M quinone or DA in 10 m M 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.
Trang 9a-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 reacfibrilliza-tion 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 (R1-NH-R2) (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
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.
Trang 10normal 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
Tokyo Chemical Industry (Tokyo, Japan)
(–)-Epigallocate-chin gallate (EGCG) was from Sigma (St Louis, MO,
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-Syn1)60
vector, and the respective proteins were expressed in
N]ammo-nium chloride Labeled or unlabeled human recombinant
a-Syn was prepared as described previously [16] The
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
(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
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
shaking for 1–24 h, followed by centrifugation at 16 000 g
for 5 min, and then aliquots of the reaction mixture were
The molecular masses of the fractions were determined by electrospray (Finnigan-LCQ-Classic, San Jose, CA, USA)
or MALDI-TOF (Bruker, Bremen, Germany) mass
also determined as a control
Redox-cycling staining a-Syn (200 lm) was incubated
Proteins were transferred from the polyacrylamide gel onto
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
(300 lm) in a buffer (10 mm phosphate, 10 mm NaCl,