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Originally published In Press as doi:10.1074/jbc.M408906200 on September 9, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47746-47753, November 12, 2004
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Functional Consequences of {alpha}-Synuclein Tyrosine Nitration

DIMINISHED BINDING TO LIPID VESICLES AND INCREASED FIBRIL FORMATION*

Roberto Hodara{ddagger}, Erin H. Norris§, Benoit I. Giasson¶, Amanda J. Mishizen-Eberz||, David R. Lynch||, Virginia M.-Y. Lee§, and Harry Ischiropoulos{ddagger}**

From the {ddagger}Stokes Research Institute and Department of Biochemistry and Biophysics, Children's Hospital of Philadelphia and the University of Pennsylvania, §Center for Neurodegenerative Disease Research and Department of Pathology and Laboratory Medicine, the University of Pennsylvania, Department of Pharmacology, the University of Pennsylvania, and ||Department of Neurology and Pediatrics, Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, August 4, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown the presence of nitrated {alpha}-synuclein ({alpha}-syn) in human Lewy bodies and other {alpha}-syn inclusions. Herein, the effects of tyrosine nitration on {alpha}-syn fibril formation, lipid binding, chaperone-like function, and proteolytic degradation were systematically examined by employing chromatographically isolated nitrated monomeric, dimeric, and oligomeric {alpha}-syn. Nitrated {alpha}-syn monomers and dimers but not oligomers accelerated the rate of fibril formation of unmodified {alpha}-syn when present at low concentrations. Immunoelectron microscopy revealed that nitrated monomers and dimers are incorporated into the fibrils. However, the purified nitrated {alpha}-syn monomer by itself was unable to form fibrils. Nitration of the tyrosine residue at position 39 was largely responsible for decreased binding of nitrated monomeric {alpha}-syn to synthetic vesicles, which correlated with an impairment of the nitrated protein to adopt {alpha}-helical conformation in the presence of liposomes. The chaperone-like activity of {alpha}-syn was not inhibited by nitration or oxidation. Furthermore, the 20 S proteasome and calpain I degraded nitrated monomeric {alpha}-syn, although at a slower rate compared with control {alpha}-syn. Collectively, these data suggest that post-translational modification of {alpha}-syn by nitration can promote the formation of intracytoplasmic inclusions that constitute the hallmark of Parkinson disease and other synucleinopathies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}-Synuclein ({alpha}-syn)1 is a 140-amino acid, natively unfolded, heat-stable, and soluble protein that is localized in the presynaptic terminals of neurons in the central nervous system (1–4), where it may regulate the release of a reserved pool of synaptic vesicles (5). {alpha}-syn interacts with a number of proteins affecting the activities of some enzymes such as phospholipase D2 (6) and tyrosine hydroxylase (7), and it can function as a chaper-one-like protein (8–10). However, under pathological conditions {alpha}-syn can aggregate into intracellular proteinaceous inclusions such as Lewy bodies (LBs) and Lewy neurites found in the brains of patients with Parkinson disease and other related disorders (11–14). Immunoelectron microscopy and thioflavin S fluorescence of LBs has revealed the presence of {alpha}-syn fibrils, indicating the ability of the protein to undergo organized fibril assembly (11–13). The formation of {alpha}-syn fibrils has been extensively studied in vitro, confirming the formation of dimers, oligomers, protofibrillar structures, and mature linear fibers (15–19), although the precise sequence of events that leads to protein aggregation in vivo is not well defined.

Post-translational modifications of {alpha}-syn may be responsible for the formation of proteinaceous inclusions. Antibodies that specifically recognize tyrosine-nitrated epitopes in {alpha}-syn decorate {alpha}-syn fibrils in LBs and Lewy neurites (20). Although these observations indicate that {alpha}-syn is a target for reactive nitrogen species in vivo, it remains unclear whether this post-translational modification is a primary event that leads to aggregation of {alpha}-syn or whether it occurs upon the reaction of reactive nitrogen species with preformed fibrils. Data gathered in simple in vitro systems appear to indicate that the latter is more likely because exposure of {alpha}-syn to nitrating agents such as peroxynitrite, myeloperoxidase plus hydrogen peroxide and nitrite, or tetranitromethane inhibit fibril formation (21, 22). However, these results appear to be in conflict with previous data indicating that the same chemical treatments result in the formation of {alpha}-syn dimers via dityrosine cross-linking, and these dimers readily serve as seeds to initiate the process of fibril formation (15). Electron microscopy data gathered in a cellular model system indicate that {alpha}-syn inclusions formed upon exposure of cells to nitrating oxidants contain fibrils (23). Moreover, these intracellular inclusions are decorated with specific monoclonal antibodies that recognize nitrated {alpha}-syn, and tyrosine residues are essential for the formation of these inclusions as transfection with {alpha}-syn with all four tyrosine residues mutated to phenylalanine fails to generate aggregates upon exposure to nitrating species (21). In part, these uncertainties may result from the presence of multiple species such as nitrated monomers and nitrated and un-nitrated dityrosine-cross-linked dimers and oligomers, as well as methionine-oxidized species in the reaction mixture. Therefore, to evaluate the role of tyrosine nitration upon {alpha}-syn aggregation, the nitrated monomeric, dimeric, and oligomeric species were isolated after chemical treatments. By employing the purified fractions, the effect of nitration and stable cross-linked oligomers on fibril formation was evaluated. Furthermore, the effect of nitration on the putative functions of {alpha}-syn, such as binding to lipid vesicles (4, 24), chaperone-like activity (8), and proteolytic processing by the 20 S proteasome (25) and calpain I (26), was evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of {alpha}-syn—The Y39F human {alpha}-syn mutant was generated by site-directed mutagenesis as described previously (21). Wild type (WT) or Y39F {alpha}-syn cDNAs subcloned into the bacterial expression vector pRK172 were expressed in Escherichia coli BL21 (DE3), and protein expression was induced with isopropyl 1-thio-{beta}-D-galactopyranoside for 2 h before harvesting. Bacterial pellets were resuspended in high salt lysis buffer (0.75 M NaCl, 100 mM MES, pH 7.0, 1 mM EDTA) containing a mixture of protease inhibitors, heated to 100 °C for 10 min, and centrifuged at 70,000 x g for 30 min. The supernatants were dialyzed against 10 mM Tris, pH 7.5, applied to a Mono Q column, and eluted with a 0–0.5 M NaCl gradient. The proteins were further purified by gel filtration on a Superdex 200 column (Amersham Biosciences) equilibrated with phosphate-buffered saline followed by a second purification through the Mono Q column. Finally, the purified protein was dialyzed against the desired working buffer. Protein concentration was determined using the bicinchoninic acid protein assay method (Pierce) using bovine serum albumin as the standard.

Nitration and Oxidation of {alpha}-syn—Peroxynitrite was synthesized as described previously (27). {alpha}-syn (10 mg/ml) was diluted into nitration buffer (200 mM potassium phosphate, pH 7.0, 25 mM NaHCO3, and 0.1 mM diethylenetriaminepentaacetic acid), and peroxynitrite was added under vigorous vortexing in three bolus additions to achieve a final 5 M excess of the protein concentration. Nitration of {alpha}-syn with tetranitromethane was performed as described previously (28) with a protein: tetranitromethane molar ratio of 1:40. Oxidation of {alpha}-syn was achieved by incubating the protein in the presence of 1 mM H2O2 and 200 nM human myeloperoxidase and full oxidation of the four methionine residues was assessed by mass spectrometry.

Separation of Different Nitrated {alpha}-syn Species—{alpha}-syn treated with a nitrating agent was run through a Superdex 200 gel filtration column equilibrated with phosphate-buffered saline at a flow rate of 0.3 ml/min. The peaks corresponding to the nitrated monomeric, dimeric, and oligomeric {alpha}-syn species were collected in separate fractions, concentrated using 5000-Da MWCO filter tubes (Millipore), run on 12% SDS-PAGE, and analyzed with an affinity-purified polyclonal antibody against nitrotyrosine raised in our laboratory.

Assembly and Fibril Formation of {alpha}-syn—{alpha}-syn fibril assembly was performed as described previously (21). Briefly, untreated {alpha}-syn or the monomerically nitrated {alpha}-syn at a concentration of 5 mg/ml was exchanged in 100 mM sodium acetate buffer (pH 7.0) containing 0.04% sodium azide. For the seeding experiments, increasing amounts of the nitrated monomer, dimer, or oligomer were added to untreated {alpha}-syn, keeping the total protein concentration at 5 mg/ml. Samples were incubated at 37 °C for a total of 5 days with continuous shaking at 1000 rpm. Each assembly reaction was overlaid with 50 µl of mineral oil to prevent condensation of samples.

Assessment of {alpha}-syn Fibrils by Centrifugal Sedimentation—Samples were centrifuged at 100,000 x g for 20 min after assembly incubation. Supernatants and pellets were separated, SDS sample buffer was added, and the samples were heated to 100 °C for 5 min. {alpha}-syn proteins were resolved by SDS-PAGE and stained with colloidal blue. In some experiments, the pellet was washed twice with sodium acetate buffer and then subjected to Western blot analysis using a nitrotyrosine antibody.

Assessment of the Formation of Amyloidogenic Fibrils Using Thioflavin T—A filtered (0.2 µm) aqueous solution of thioflavin T (Sigma) was prepared at 10 µM in 90 mM glycine NaOH buffer (pH 8.5) prior to measurements. At various time points, aliquots of the {alpha}-syn incubation samples were diluted to 10 µM in 100 µl of water, and then 100 µlofthe 10-µM thioflavin T solution was added. Fluorescence measurements were conducted in black 96-well plates using a SpectraMax Gemini fluorometer (Molecular Devices, Sunnyvale, CA) and SoftMax Pro 4.0 software ({lambda}ex = 450 nm, {lambda}em = 490 nm).

Immunoelectron Microscopy—After assembly of {alpha}-syn into fibrils, samples were prepared for electron microscopy analysis as described previously (21). For direct inspection of {alpha}-syn fibril formation, samples were applied to 300-mesh carbon-coated copper grids and were negatively stained with 1% uranyl acetate. Samples were also analyzed using immunoelectron microscopy (IEM) techniques to determine the presence of nitrated filaments within the assembled structures. {alpha}-syn samples were applied to 300-mesh carbon-coated grids, blocked with 1% bovine serum albumin in phosphate-buffered saline, and immunostained with primary monoclonal antibody nSyn14, which specifically recognizes nitrated {alpha}-syn (20). Samples were then decorated with anti-mouse secondary antibody conjugated to 5-nm gold particles and were negatively stained with 1% uranyl acetate. A JEOL 1010 electron microscope (Peabody, MA) was used to examine these samples with magnifications up to x200,000.

Chaperone-like Activity Assay—The insulin assay was performed as described previously (8). Briefly, bovine insulin, 0.15 mg/ml in 100 mM phosphate buffer, pH 7.0, was incubated with 20 mM DTT at 25 °C in the absence or presence of unmodified, nitrated, or oxidized {alpha}-syn (50 µM). Aggregation of the insulin B chain after reduction with DTT was followed by monitoring of light scattering at 360 nm.

Binding of {alpha}-syn to Unilamellar Lipid Vesicles—Unilamellar lipid vesicles were synthesized as follows. 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphate (POPA) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids (Alabaster, AL) in specially packaged glass vials containing 25 mg sealed under argon. Phospholipids were dissolved in cyclohexane, mixed in 1:1 ratio (in the case of mixed POPA/POPC unilamellar lipid vesicles), flash-frozen in liquid nitrogen, lyophilized, and suspended in buffer A (50 mM Tris buffer, pH 7.6) using mild bath sonication for 10 min. To minimize spontaneous oxidation of lipids, all manipulations were performed under argon. Homogeneous unilamellar lipid vesicles were made using a syringe extruder (Avanti Polar Lipids) and a 100-nm filter. Phospholipid concentration was determined by phosphate assay (29, 30). POPC or POPC/POPA unilamellar lipid vesicles at a concentration of 20 mg/ml were incubated with either untreated {alpha}-syn or nitrated monomeric {alpha}-syn (at 1 mg/ml) for 2 h in buffer A. Samples were then loaded on a Superdex 200 gel filtration column, equilibrated with buffer A, and eluted using a flow rate of 0.5 ml/min following absorbance at 210 and 280 nm. The protein and lipid fractions were collected, SDS-PAGE was performed, and the fractions were stained with colloidal blue (Invitrogen). The area under the curve of the protein peaks was integrated to calculate the fraction bound to unilamellar lipid vesicles.

Circular Dichroism Spectroscopy—The circular dichroism spectra of untreated or nitrated monomeric {alpha}-syn, in the presence or absence of unilamellar lipid vesicles (2 mg/ml), were recorded using a Jasco J-810 spectropolarimeter. Spectra were collected at 25 °C in a 0.1-cm path length quartz cuvette containing the sample at concentrations of 0.1 mg/ml protein in potassium phosphate buffer (50 mM, pH 7.6).

Degradation of {alpha}-syn by the 20 S Proteasome and Calpain I—The 20 S proteasome and calpain I were from Calbiochem. Assays were performed as described previously (26, 31). Untreated or nitrated monomeric {alpha}-syn at 500 nM was incubated with 10 nM 20 S proteasome at 37 °C in buffer A (20 mM Tris-HCl, pH 7.1, 20 mM NaCl, 10 mM MgCl2, 0.25 mM ATP, 1 mM dithiothreitol (DTT)). The proteasome inhibitor, lactacystin, was used at a 50 µM concentration. For calpain I studies, {alpha}-syn protein (0.1 µg/µl) was incubated with calpain I (1 units/ml) in buffer B (40 mM HEPES, pH 7.2, and 5 mM DTT) at 37 °C, and the reaction was initiated by the addition of calcium (1 mM final). Aliquots were taken at different time points, and the reaction was stopped by the addition of SDS sample buffer and heating the sample to 100 °C for 5 min. Protein degradation was assessed by immunoblotting with the monoclonal antibody to {alpha}-syn, Syn 208 (32). Immunoblots were analyzed using NIH Image.

Mass Spectrometry Studies—Mass spectrometry was performed on an Agilent 1100 Series quadrupole mass spectrometer equipped with an electrospray ion source, and the spectrometer was operated in positive ion mode as described previously (33).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromatographic Separation of {alpha}-syn Modified Species— A major obstacle in studying the biochemical and biophysical properties of nitrated {alpha}-syn is obtaining a homogeneous preparation of modified protein. Therefore, after the reaction of recombinant {alpha}-syn with nitrating agents such as peroxynitrite or tetranitromethane, the resulting {alpha}-syn nitrated monomers as well as nitrated dimers covalently linked by the formation of dityrosine and higher molecular mass oligomers were separated by gel filtration chromatography (Fig. 1). The chromatographic elution profile and the corresponding SDS-PAGE and Western blot analyses of the isolated fractions (Fig. 1) indicate that the different species were successfully separated with minimal cross-contamination. Mass spectrometric analysis of the purified nitrated monomer revealed the presence of mono-, di-, and tri-nitrated species. Digestion with trypsin and cyanogen bromide followed by liquid chromatography-mass spectrometry analysis of peptides revealed that all four tyrosine residues could potentially be nitrated (data not shown and 31). {alpha}-syn proteins nitrated at Tyr-39 as well as at Tyr-125, Tyr-133, or Tyr-136 have been detected in LBs of human brains affected with Parkinson disease (20).



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  		FIG. 1.
A, separation of nitrated {alpha}-syn oligomers (O), nitrated dimers (D), and nitrated monomers (M) by gel filtration chromatography. mAU, absorbance units x 10–3. B, SDS-polyacrylamide gel of the monomeric fraction (lane 1) compared with the whole reaction mixture (lane 2) as seen by colloidal blue stain. C, Western blot analyzed with an affinity-purified polyclonal anti-nitrotyrosine antibody shows separation of fractions and nitration. Lane 1, unmodified {alpha}-syn; lane 2, monomers; lane 3, dimers; lane 4, oligomers; and lane 5, the entire reaction mixture.

 
Effect of Nitration on {alpha}-syn Fibril Formation—Purified tyrosine-nitrated {alpha}-syn monomers were unable to assemble into fibrils as assessed by the thioflavin T assay and sedimentation (Fig. 2, A and C, respectively). These results are consistent with previous observations (21, 22). However, when unmodified {alpha}-syn was co-incubated with increasing amounts of the tyrosine-nitrated {alpha}-syn monomers in ratios of 1:10, 1:5, and 1:1, a significant change in the kinetics of fibril formation was observed. Specifically, a shorter lag phase and an increase in the velocity of fibril assembly and intensity of thioflavin T fluorescence were observed (Fig. 2A). Furthermore, extensive washing of the protein in the pellet fraction followed by blotting with anti-nitrotyrosine antibodies revealed that nitrated monomers are present in the pellet fraction after 96 h of incubation (Fig. 2D). However, the 1:1 mixture showed lower thioflavin T fluorescence intensity (Fig. 2A) despite the presence of {alpha}-syn in the pellets after sedimentation analysis (Fig. 2C) and the presence of fibrils upon IEM evaluation (Fig. 3). To explain this apparent discrepancy, the interference of free 3-nitrotyrosine with thioflavin T fluorescence was examined (Fig 2B). At the pH used in the thioflavin T assay, 3-nitrotyrosine shows a maximal absorption at 422 nm, close to the wavelength used to excite thioflavin T (450 nm). Because the liquid chromatography-mass spectrometry data indicated that at least two nitrated tyrosine residues were present per {alpha}-syn molecule, the 1:5 and 1:1 samples represent ~70 and 350 µM 3-nitrotyrosine, respectively. Fig. 2B shows that thioflavin T fluorescence of preformed {alpha}-syn fibrils was markedly inhibited in the presence of 350 µM free 3-nitrotyrosine, whereas 70 µM had minimal effect. These data indicate that the loss of thioflavin T fluorescence observed in the 1:1 ratio is due to fluorescence quenching by 3-nitrotyrosine.



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  		FIG. 2.
A, kinetic analysis of thioflavin T fluorescence of unmodified {alpha}-syn (ctl), purified nitrated monomer (M alone), mixtures of 1:10, 1:5, and 1:1 nitrated {alpha}-syn monomers with unmodified {alpha}-syn (M1:10, M1:5, and M1:1), mixture of 1:10 of nitrated {alpha}-syn dimers (D1:10), and nitrated oligomers (O1:10) with unmodified {alpha}-syn. B, unmodified {alpha}-syn was incubated for 96 h, and thioflavin T fluorescence was then recorded in the presence of increasing concentrations of free 3-nitrotyrosine. C, after incubation for 96 h, samples from A were centrifuged at 100,000 x g, and the resulting supernatant (S) and pellet (P) fractions were separated and resolved by SDS-PAGE and stained with colloidal blue. Olig, oligomer; Di, dimer. D, after incubation for 96 h, samples were centrifuged at 100,000 x g and washed extensively. An affinity-purified anti-nitrotyrosine antibody was used to detect nitration in supernatant and pellets of these samples. Representative gels and blots are from triplicate determinations. AFU, arbitrary fluorescence units.

 



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  		FIG. 3.
Immunoelectron microscopy using mouse monoclonal antibody nSyn14 against nitrated {alpha}-syn. Unmodified {alpha}-syn showed no gold labeling, serving as a negative control (A). Positive staining for nitrated {alpha}-syn monomers was observed along the fibrils as shown in the 1:10 and 1:1 ratio of nitrated monomers to unmodified {alpha}-syn micrographs (B and C, respectively, gold labeling indicated by arrowheads). Nitrated {alpha}-syn dimers were also observed along the fibrils (D) at a ratio of 1:10 nitrated dimers to unmodified {alpha}-syn. Scale bar, 100 nm.

 
Co-incubation of unmodified {alpha}-syn with nitrated {alpha}-syn dimers in a 1:10 ratio significantly accelerates the rate of fibril formation and markedly increases total fluorescence (Fig. 2A). However, the sedimentation assay does not show evidence of a significant increase of {alpha}-syn in the pellet fraction as compared with unmodified {alpha}-syn incubated alone or after co-incubations of unmodified {alpha}-syn with a nitrated {alpha}-syn monomer (Fig. 2C). These results imply that nitrated dimers promote a more organized assembly of {alpha}-syn into filaments rich in {beta}-sheet conformation. Interestingly, agitation for 96 h resulted in the formation of {alpha}-syn dimers (Fig. 2C), which were not present before incubation either in the unmodified {alpha}-syn preparation (not shown) or in the purified nitrated {alpha}-syn monomer fraction (Fig. 1). These dimers appeared in the pellet fraction, with the exception of the nitrated {alpha}-syn monomer fraction incubated alone, where they remained soluble (Fig. 2C).

The co-incubation of nitrated oligomers with unmodified {alpha}-syn inhibited both thioflavin T fluorescence and the formation of a protein pellet after sedimentation (Fig. 2, A and C). Under these conditions, electron microscopy analysis failed to detect fibrils (data not shown). Because the fraction of oligomeric {alpha}-syn contains some dimers (Fig. 1C), these data suggest that oligomeric {alpha}-syn species have a predominant effect in inhibiting fibril formation.

IEM Analysis—To confirm the data from the thioflavin T and sedimentation studies, we performed IEM analyses. These studies revealed the presence of monomerically nitrated {alpha}-syn in fibrils composed of unmodified {alpha}-syn (Fig. 3, B and C). Similarly, nitrated {alpha}-syn dimers incorporate into fibrils as revealed by the presence of gold labeling along the fibrils (Fig. 3D).

Nitrated {alpha}-syn Monomer Shows Decreased Binding to Lipid Vesicles—The interaction of {alpha}-syn with lipid vesicles is believed to play a role in regulation of neurotransmission at presynaptic terminals. Binding preferably occurs to vesicles containing acidic phospholipids, which leads to a significant increase in {alpha}-helical conformation of {alpha}-syn (4). Purified nitrated {alpha}-syn monomers were incubated with POPC or a mixture of POPC/POPA unilamellar lipid vesicles at room temperature for 2 h in a 20:1 (lipid:protein) mass ratio, and binding was studied by gel filtration chromatography. In agreement with previous results, SDS-PAGE analyses of the fractions eluted from the column revealed that neither unmodified {alpha}-syn nor the purified nitrated {alpha}-syn monomer binds to POPC vesicles (Fig. 4A). However, when incubated with POPC/POPA vesicles, 85% of unmodified {alpha}-syn eluted with the vesicle fraction, whereas the remaining unbound protein was below the detection limit of colloidal blue staining (Fig. 4, A and B). In contrast, a significant amount of nitrated {alpha}-syn monomers were detected in the unbound fraction (Fig. 4A). Only 55% of nitrated {alpha}-syn binds to POPC/POPA vesicles (Fig. 4B). Because the N-terminal segment of the protein is responsible for binding to vesicles (4), we employed the Tyr-39 to phenylalanine-mutated {alpha}-syn (Y39F) to investigate the functional consequence of nitration of this residue. The unmodified Y39F mutant showed the same extent of binding to POPC/POPA vesicles as the unmodified WT protein (Fig. 4B). As expected, the nitrated Y39F mutant (nitrated on the three tyrosine residues in the C-terminal domain) showed significant binding (70%) to POPC/POPA vesicles as compared with the nitrated WT monomer (Fig. 4B).



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  		FIG. 4.
Binding of {alpha}-syn to lipid vesicles. A, SDS-polyacrylamide gel stained with colloidal blue shows the unmodified (Syn) and nitrated monomer (M) {alpha}-syn fractions bound (B) or unbound (U) to lipid vesicles. B, quantification (n = 3) of vesicular binding of the unmodified wild type (WT) and unmodified mutant (Y39F) {alpha}-syn and their respective nitrated monomers (WT-M and Y39F-M). *, p < 0.05 difference as compared with unmodified {alpha}-syn; #, p < 0.05 as compared with wild type nitrated monomers. C and D, CD spectra of unmodified wild type and mutant {alpha}-syn and their respective nitrated monomers in the absence (C) or presence (D) of POPC/POPA vesicles.

 
To evaluate whether nitration changes the secondary structure of {alpha}-syn, leading to decreased binding to acidic unilamellar lipid vesicles, CD spectra of nitrated {alpha}-syn monomers in the absence or presence of unilamellar lipid vesicles were obtained (Fig. 4, C and D). Nitration of WT {alpha}-syn resulted in a significant change in its secondary structure in the absence of vesicles (Fig. 4C). Data showed a strong negative band at 200 nm, indicating a more unordered conformation. The Y39F mutant, however, did not show this effect after nitration (Fig. 4C). In agreement with previous studies (4), incubation of unmodified {alpha}-syn with mixed POPC/POPA vesicles increased the {alpha}-helical content (Fig. 4D). Unmodified Y39F {alpha}-syn assumed the same extent of {alpha}-helical conformation as unmodified WT {alpha}-syn in the presence of vesicles. The nitrated WT monomers showed a decrease in the ability to adopt {alpha}-helical conformation in the presence of vesicles, characterized by the decreased molar ellipticity at 208 and 222 nm in the CD spectra (Fig. 4D). Y39F {alpha}-syn showed only marginal changes of these bands after nitration (Fig. 4D). These data indicate that nitration of {alpha}-syn at tyrosine residue 39 is largely responsible for the inability of the nitrated monomer to bind to vesicles.

Nitration or Oxidation Does Not Alter the Chaperone-like Activity of {alpha}-syn—Previous studies showed that {alpha}-syn interacts stoichiometrically with unfolded proteins and, in a chaperone-like fashion, prevents the aggregation of these proteins (8–10). This function may be related to the highly polar, flexible, and unstructured C-terminal domain, a feature shared by chaper-one proteins such as HSP25 and {alpha}-crystallin (8–10, 34). This C-terminal domain contains three of the four tyrosine residues in {alpha}-syn, and these three residues are modified by nitration, albeit at different levels, in human lesions (20). The chaperone activity of {alpha}-syn was evaluated by the ability of the protein to prevent the aggregation of the insulin B-chain after reduction with DTT (8). RNase A, a protein with similar molecular weight to {alpha}-syn without chaperone activity, was used as a negative control, and HSP27 was used as a positive control. Nitrated {alpha}-syn monomers prevented the DTT-induced unfolding and aggregation of the insulin B-chain to the same extent as an equal concentration of unmodified {alpha}-syn (Fig. 5). Similarly, oxidation of {alpha}-syn by hydrogen peroxide plus myeloperoxidase had no effect on its chaperone-like activity (Fig. 5).



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  		FIG. 5.
Aggregation of the insulin B chain after reduction with DTT was followed by monitoring light scattering at 360 nm. Insulin aggregation was performed in the absence (control) or presence of 50 µM unmodified ({alpha}-Syn), nitrated ({alpha}-Syn-NO2), or oxidized ({alpha}-Syn-Ox) {alpha}-syn. HSP27 was used as a positive control, and RNase A was used as a negative control. These data are from a representative experiment of a triplicate determination. Abs., absorbance.

 
Proteolytic Degradation of Nitrated {alpha}-syn by the Proteasome and Calpain I—Although the 26 S and 20 S proteasome (25, 31, 35–37), lysosomes (23, 37, 38), and calpain I (26, 39) have been implicated in proteolytic degradation of {alpha}-syn, it is not clearly established which pathway is responsible for the turnover. Purified 20 S proteasome appeared to degrade nitrated {alpha}-syn monomers more slowly than the unmodified WT protein (Fig. 6, A and B). Although unmodified {alpha}-syn was completely degraded by 10 min, full-length {alpha}-syn (~16 kDa) was still apparent at 20 min in the monomerically nitrated protein (Fig. 6A). Densitometric analysis of three similar experiments showed full-length nitrated {alpha}-syn still remaining at 30 min (Fig. 6A). Similarly, calpain I degradation of nitrated {alpha}-syn also occurred at a slower rate compared with control {alpha}-syn (Fig. 6B). For both assays, the pattern of degradation appears to be identical for both nitrated and unmodified {alpha}-syn (Fig. 6).



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  		FIG. 6.
Representative blots showing a time course for the degradation of control and nitrated protein by purified 20 S proteasome (A and B) or calpain I (C and D). The bar graphs represent densitometric analysis from three independent experiments. Syn, unmodified {alpha}-syn; M, nitrated {alpha}-syn monomer; Lac, lactacystin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chemical and metabolic modifiers have been shown to alter the kinetics of {alpha}-syn fibril formation as well as the biophysical properties of the fibrils (21, 22, 33, 40–45). One of the chemical modifiers, tyrosine nitration, has emerged as a relevant post-translational modification of {alpha}-syn, because it has been shown to occur in vivo in several human pathological conditions (20). However, in vitro studies have failed to provide conclusive evidence for a functional role of tyrosine nitration on {alpha}-syn fibril formation in part because these studies have employed the entire reaction mixture that results from exposure to nitrating agents as the starting material (21, 22). These mixtures contain dityrosine-cross-linked {alpha}-syn dimers and oligomers in addition to nitrated monomers that may have profound yet opposite effects on the process of fibril formation (15, 46). Herein, we successfully separated these species using gel filtration chromatography into three fractions containing nitrated monomers, dimers, and the higher molecular mass oligomers (Fig. 1). Using these fractions, the data corroborated previous findings that nitrated {alpha}-syn is incapable of forming fibrils (21, 22). However, co-incubation experiments with unmodified {alpha}-syn revealed that the nitrated monomers are not only incorporated into fibrils of unmodified {alpha}-syn (as assessed by IEM) but also appear to increase the rate of fibril formation when present at low concentrations. These co-incubation experiments are relevant because in vivo the ratio of nitrated {alpha}-syn to unmodified {alpha}-syn is expected to be low, at least in the initial phases of the disease.

Interestingly, nitrated {alpha}-syn dimers and the nitrated oligomers appear to have completely opposite effects on the fibrillization of unmodified {alpha}-syn. Spontaneously formed {alpha}-syn dimers as well as dimers formed after exposure to peroxynitrite provide the critical rate-limiting step in the nucleation of {alpha}-syn fibrils (15). Consistent with these observations, nitrated dimers generated by exposure to peroxynitrite or other nitrating species significantly promote fibril formation (Fig. 2). However, oligomers of higher molecular mass inhibited fibril formation, even in the presence of some dimers. Therefore, it is likely that in previous studies, the presence of oligomers was partially responsible for the inhibition of fibril formation. It is also possible that these oligomeric species may constitute the distinct octamers reported previously upon nitration of the protein (22).

Although {alpha}-syn protofibrils may disrupt and permeabilize synthetic lipid vesicles (47–49), it has been proposed that the monomeric form can stabilize presynaptic vesicles by binding to defects in the membrane bilayer (50). Herein, we show that nitration of {alpha}-syn hinders its ability to bind to lipid vesicles, an effect mediated largely by nitration of Tyr-39 (as expected by its location in the N terminus of the {alpha}-syn sequence). The addition of the rather bulky nitro group at the ortho position of the aromatic ring not only induces a significant shift in the pKa of the tyrosine residue (from 10.01 to 7.2), thus adding a negative charge, but may also prevent the rotational ability of the tyrosine residue, all of which may destabilize the {alpha}-helical conformation induced upon binding to lipid vesicles. Interestingly, in this regard, nitrated {alpha}-syn behaves similarly to the A30P {alpha}-syn, as this pathogenic mutant also shows a profound inhibition in its ability to bind to lipid vesicles (51). These findings could potentially explain {alpha}-syn toxicity, because membrane-bound {alpha}-syn shows less propensity to form fibrils (52, 53), and a nitrating insult to the cell would result in an increase in the unbound {alpha}-syn fraction that promotes the formation of intracellular aggregates. As shown in Fig. 6, the soluble nitrated {alpha}-syn pool would not be as efficiently cleared by intracellular proteases as unmodified {alpha}-syn, resulting in a prolonged half-life that may potentiate its ability to accelerate the formation of fibrils (Fig. 2). Calpain I (26, 39) and perturbations in the ubiquitin-proteasome (31, 37, 54, 55) system have been implicated in the pathogenesis of Parkinson disease and related pathologies. It seems plausible, therefore, that nitration of {alpha}-syn could disturb the balance between lipid binding and removal of the modified protein in favor of its accumulation and aggregation in the cytosol.

Another putative function that has been assigned to {alpha}-syn is its chaperone-like activity, an effect entirely mediated through the C-terminal domain of the protein (8–10). Unlike the nitration of Tyr-39, which significantly inhibits the association of {alpha}-syn with a lipid vesicle, nitration of tyrosine residues located in the C terminus (Tyr-125, Tyr-133, and Tyr-136) had no effect on the chaperone-like activity of {alpha}-syn. Similarly, oxidation of methionine residues with H2O2 and myeloperoxidase had no effect on this function.

Collectively, the data suggest that the presence of nitrated {alpha}-syn in LBs and other proteinaceous inclusions may not be an epiphenomenon of formation of nitrating species; rather it may signify a post-translational modification that reduces its association with lipid vesicles, prolonging its intracellular half-life and promoting self-aggregation and formation of {alpha}-syn inclusions.


   FOOTNOTES
 
* This work was supported by grants from the NIA, National Institutes of Health (to D. R. L., V. M.-Y. L., and H. I.), a Pioneer Award from the Alzheimer's Association (to V. M.-Y. L.), and an AME-APDA fellowship (to D. R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Stokes Research Inst., Children's Hospital of Philadelphia, 416D Abramson Research Center, 34th St. and Civic Center Blvd. Philadelphia, PA 19104-4318. Tel.: 215-590-5320; Fax: 215-590-4267; E-mail: ischirop{at}mail.med.upenn.edu.

1 The abbreviations used are: {alpha}-syn, {alpha}-synuclein; LBs, Lewy bodies; WT, wild type; IEM, immunoelectron microscopy; DTT, dithiothreitol; POPA, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphate; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank the Biochemical Imaging Core Facility of the University of Pennsylvania for assistance with the electron microscopy and Dr. Ian Murray for help with lipid vesicle preparation.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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