Peroxidase Mechanism of Lipid-dependent Cross-linking of Synuclein with Cytochrome c

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that α-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H2O2. Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage. Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that α-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H2O2. Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage. Lewy bodies (LBs), 3The abbreviations used are:LBLewy bodySynsynucleincyt ccytochrome cLNLewy neuritisPDParkinson diseaseDAdopamineCLcardiolipinTOCLtetraoleoyl-CLTLCLtetralinoleoyl-CLPIphosphatidylinositolPAphosphatidic acidPCphosphatidylcholinePSphosphatidylserineNAO10-N-nonyl acridine orangeDMPO5,5-dimethyl-1-pyrroline-N-oxideMECmouse embryonic cellsActDactinomycin Dt-BuOOHtert-butyl hydroperoxideDOPCdioleoylphosphatidylcholineDTPAdiethylenetriaminepentaacetic acidDTTdithiothreitolMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightLC-ESI-MSliquid chromatography-electrospray ionization-tandem mass spectrometryHPLChigh performance liquid chromatographyDMSOdimethyl sulfoxideNBD-CL1,1′,2-trioleoyl-2′-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-cardiolipinFRETfluorescence resonance energy transfersiRNAsmall interfering RNAFBSfetal bovine serum. 3The abbreviations used are:LBLewy bodySynsynucleincyt ccytochrome cLNLewy neuritisPDParkinson diseaseDAdopamineCLcardiolipinTOCLtetraoleoyl-CLTLCLtetralinoleoyl-CLPIphosphatidylinositolPAphosphatidic acidPCphosphatidylcholinePSphosphatidylserineNAO10-N-nonyl acridine orangeDMPO5,5-dimethyl-1-pyrroline-N-oxideMECmouse embryonic cellsActDactinomycin Dt-BuOOHtert-butyl hydroperoxideDOPCdioleoylphosphatidylcholineDTPAdiethylenetriaminepentaacetic acidDTTdithiothreitolMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightLC-ESI-MSliquid chromatography-electrospray ionization-tandem mass spectrometryHPLChigh performance liquid chromatographyDMSOdimethyl sulfoxideNBD-CL1,1′,2-trioleoyl-2′-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-cardiolipinFRETfluorescence resonance energy transfersiRNAsmall interfering RNAFBSfetal bovine serum. mitochondrial impairment, and oxidative stress are cardinal features of Parkinson disease (PD) and several related neurodegenerative disorders (1.Betarbet R. Sherer T.B. Di Monte D.A. Greenamyre J.T. Brain Pathol. 2002; 12: 499-510Crossref PubMed Scopus (122) Google Scholar, 2.Irizarry M.C. Growdon W. Gomez-Isla T. Newell K. George J.M. Clayton D.F. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 334-337Crossref PubMed Scopus (355) Google Scholar). Aggregation of α-synuclein (Syn), an abundant protein in synaptic terminals, plays a major role in the formation of LBs (3.Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 4.Dickson D.W. Curr. Opin. Neurol. 2001; 14: 423-432Crossref PubMed Scopus (152) Google Scholar). Neither the mechanisms of LB production nor their pathogenic or protective roles in neurodegeneration are well understood. Lewy body synuclein cytochrome c Lewy neuritis Parkinson disease dopamine cardiolipin tetraoleoyl-CL tetralinoleoyl-CL phosphatidylinositol phosphatidic acid phosphatidylcholine phosphatidylserine 10-N-nonyl acridine orange 5,5-dimethyl-1-pyrroline-N-oxide mouse embryonic cells actinomycin D tert-butyl hydroperoxide dioleoylphosphatidylcholine diethylenetriaminepentaacetic acid dithiothreitol matrix-assisted laser desorption/ionization time-of-flight liquid chromatography-electrospray ionization-tandem mass spectrometry high performance liquid chromatography dimethyl sulfoxide 1,1′,2-trioleoyl-2′-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-cardiolipin fluorescence resonance energy transfer small interfering RNA fetal bovine serum. Lewy body synuclein cytochrome c Lewy neuritis Parkinson disease dopamine cardiolipin tetraoleoyl-CL tetralinoleoyl-CL phosphatidylinositol phosphatidic acid phosphatidylcholine phosphatidylserine 10-N-nonyl acridine orange 5,5-dimethyl-1-pyrroline-N-oxide mouse embryonic cells actinomycin D tert-butyl hydroperoxide dioleoylphosphatidylcholine diethylenetriaminepentaacetic acid dithiothreitol matrix-assisted laser desorption/ionization time-of-flight liquid chromatography-electrospray ionization-tandem mass spectrometry high performance liquid chromatography dimethyl sulfoxide 1,1′,2-trioleoyl-2′-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-cardiolipin fluorescence resonance energy transfer small interfering RNA fetal bovine serum. In nigrostriatal dopaminergic synaptic terminals, mitochondria, harboring a host of death-initiating factors, are in peril of damage by reactive oxygen species generated by disrupted electron transport and/or oxidative metabolism of dopamine (DA). Because cytochrome c (cyt c)-dependent formation of apoptosomes and activation of caspases designates a point of no return in the apoptotic program, release of proapoptotic factors from synaptic mitochondria could threaten the integrity of the entire neuron. How neurons protect themselves against inadvertent release of death signals from damaged synaptic mitochondria is not known. The N-terminal fragment of Syn contains six variants of an 11-amino acid consensus motif that include an apolipoprotein-like class A2 helix participating in binding of different lipids, particularly anionic phospholipids (5.Shults C.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1661-1668Crossref PubMed Scopus (343) Google Scholar). This domain is believed to be important for Syn functions in regulation of neuronal lipid metabolism, particularly turnover of a mitochondria-specific phospholipid, cardiolipin (CL) (6.Ellis C.E. Murphy E.J. Mitchell D.C. Golovko M.Y. Scaglia F. Barcelo-Coblijn G.C. Nussbaum R.L. Mol. Cell. Biol. 2005; 25: 10190-10201Crossref PubMed Scopus (203) Google Scholar). However, the relevance of the Syn lipid binding capacity in regulating neuronal injury (apoptotic) responses has not been established. It is believed that oxidative stress participates in the accumulation of LB and Lewy neurites (LN) through yet to be identified pathways (7.Giasson B.I. Duda J.E. Murray I.V. Chen Q. Souza J.M. Hurtig H.I. Ischiropoulos H. Trojanowski J.Q. Lee V.M. Science. 2000; 290: 985-989Crossref PubMed Scopus (1354) Google Scholar). Reportedly, Syn is co-localized with cyt c in LBs (8.Hashimoto M. Takeda A. Hsu L.J. Takenouchi T. Masliah E. J. Biol. Chem. 1999; 274: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), indicating a potential interaction between the two proteins. Because cyt c is a redox-active hemeprotein (9.Burkitt M. Jones C. Lawrence A. Wardman P. Biochem. Soc. Symp. 2004; 71: 97-106Crossref PubMed Scopus (15) Google Scholar, 10.Qian S.Y. Chen Y.R. Deterding L.J. Fann Y.C. Chignell C.F. Tomer K.B. Mason R.P. Biochem. J. 2002; 363: 281-288Crossref PubMed Google Scholar), its presence in the LBs in conjunction with Syn may also provide a mechanistic link of LBs with oxidative stress. We have recently reported that cyt c interacts with CL in mitochondria early in apoptosis and with phosphatidylserine (PS) in the plasma membrane after its release into the cytosol (11.Jiang J. Kini V. Belikova N. Serinkan B.F. Borisenko G.G. Tyurina Y.Y. Tyurin V.A. Kagan V.E. Lipids. 2004; 39: 1133-1142Crossref PubMed Scopus (34) Google Scholar, 12.Kagan V.E. Tyurin V.A. Jiang J. Tyurina Y.Y. Ritov V.B. Amoscato A.A. Osipov A.N. Belikova N.A. Kapralov A.A. Kini V. Vlasova I.I. Zhao Q. Zou M. Di P. Svistunenko D.A. Kurnikov I.V. Borisenko G.G. Nat. Chem. Biol. 2005; 1: 223-232Crossref PubMed Scopus (951) Google Scholar). In both cases, this results in redox activation of cyt c and the production of complexes with high peroxidase activity that effectively catalyze peroxidation of the respective phospholipids (13.Bayır H. Fadeel B. Palladino M. Witasp E. Kurnikov I. Tyurina Y.Y. Tyurin V.A. Amoscato A. Jiang J. Kochanek P.M. DeKosky S. Greenberger J. Shvedova A.A. Kagan V.E. Biochim. Biophys. Acta. 2006; 1757: 648-659Crossref PubMed Scopus (151) Google Scholar). Based on these facts, we hypothesize and provide experimental evidence that Syn acts as a sacrificial scavenger of cytosolic cyt c inadvertently released from synaptic mitochondria to prevent its migration into the soma, i.e. spread of the proapoptotic signal and cell death. This vital function is realized through the emergence of a peroxidase activity of the cyt c-Syn-phospholipid complex that cross-links its components and yields covalently conjugated protein-lipid hetero-oligomers. The latter maintain lingering peroxidase activity. Thus protection against acute apoptotic cell death comes with a penalty of Syn-cyt c aggregation into a peroxidase complex capable of inducing protracted oxidative stress. Our results present a novel biochemical mechanism likely involved in Lewy body formation and explain a known paradox of a dual protective and deleterious role that Syn plays in neuronal cells. HeLa, HL-60, and SH-SY5Y cells were purchased from the American Type Culture Collection and cultured in 1:1 mixture of Eagle's minimum essential medium and Ham's F-12 medium supplemented with 10% of fetal bovine serum (FBS), 1.5 g/liter sodium bicarbonate, 2 mm l-glutamine, 0.5 mm sodium pyruvate, and 0.05 mm nonessential amino acids. For apoptosis induction, HeLa cells were incubated with tert-butyl hydroperoxide (t-BuOOH) (400 μm) or ActD (200 ng/ml) for 16 h; SH-SY5Y cells were incubated with t-BuOOH (10 μm for 16 h) or ActD (10 μg/ml for 18 h). At the end of incubation, the attached cells were harvested by trypsinization and pooled with the detached cells from supernatant for PS externalization analysis and immunostaining. Cyt c-deficient HeLa cells were generated using siRNA-expressing plasmid (pSEC hygro vector, Ambion) as described previously (12.Kagan V.E. Tyurin V.A. Jiang J. Tyurina Y.Y. Ritov V.B. Amoscato A.A. Osipov A.N. Belikova N.A. Kapralov A.A. Kini V. Vlasova I.I. Zhao Q. Zou M. Di P. Svistunenko D.A. Kurnikov I.V. Borisenko G.G. Nat. Chem. Biol. 2005; 1: 223-232Crossref PubMed Scopus (951) Google Scholar) and cultured in Dulbecco's modified Eagle's medium supplemented with 15% FBS, 25 mm HEPES, 50 mg/liter uridine, 110 mg/liter pyruvate, 2 mm glutamine, 1× nonessential amino acids, 0.05 mm 2′-mercaptoethanol. Syn knockdown HeLa cells were generated using siRNA-expressing plasmid (pSEC hygro vector, Ambion). Target sequence (AAGAGGGTGTTCTCTATGTAG) was cloned into pSEC hygro vector, and the resultant plasmid was transfected into HeLa cells. Positive clones were selected by hygromycin. Mouse embryonic cells (MECs) (courtesy of X. Wang, University of Texas, Dallas) were derived from 8- to 9-day-old mouse embryos by Li et al. (14.Li K. Li Y. Shelton J.M. Richardson J.A. Spencer E. Chen Z.J. Wang X. Williams R.S. Cell. 2000; 101: 389-399Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). MECs were cultured in Dulbecco's modified Eagle's medium supplemented with 15% FBS, 25 mm HEPES, 50 mg/liter uridine, 110 mg /liter pyruvate, 2 mm glutamine, 1× nonessential amino acids, 0.05 mm 2′-mercaptoethanol, 0.5 × 106 units/liter mouse leukemia inhibitory factor. Syn protein was delivered into cells using Chariot (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 0.03 × 106/well in a 24-well plate and allowed to attach overnight. Chariot-Syn complex (2 μl, 0.5 μg) was incubated with cells for 3 h for integration. After that, cells were treated with 50 ng/ml ActD for 18 h. At the end of incubation, attached cells were harvested by trypsinization and pooled with detached cells from supernatant. Caspase-3/7 activity was determined using a caspase-3/7 Glo kit (Promega, San Luis Obispo, CA). Liposomes containing dioleoyl-phosphatidylcholine (DOPC) and tetraoleoyl-CL (TOCL) (or other anionic lipids) (lipid/DOPC ratio 1:1), were prepared in 20 mm HEPES, pH 7.4, by sonication under N2 and used immediately after preparation. To prevent redox cycling with free metals, diethylenetriaminepentaacetic acid (DTPA) (100 μm) was added to all solutions used. Fibrillated (aged) Syn was prepared by incubation of wild-type Syn and its mutants (200 μm) in 20 mm HEPES, 100 μm DTPA, pH 7.4, with shaking at 200 rpm for 6 days at 37 °C. Mitochondria were isolated as described previously (12.Kagan V.E. Tyurin V.A. Jiang J. Tyurina Y.Y. Ritov V.B. Amoscato A.A. Osipov A.N. Belikova N.A. Kapralov A.A. Kini V. Vlasova I.I. Zhao Q. Zou M. Di P. Svistunenko D.A. Kurnikov I.V. Borisenko G.G. Nat. Chem. Biol. 2005; 1: 223-232Crossref PubMed Scopus (951) Google Scholar). Briefly, harvested cells were resuspended in isolation buffer containing 300 mm mannitol, 10 mm HEPES-KOH, pH 7.4, 0.2 mm EDTA, 0.1% bovine serum albumin, and protease inhibitor mixture (Roche Applied Science) homogenized on ice with a glass homogenizer, and then centrifuged at 1000 × g for 10 min at 4 °C. The resulting supernatants were centrifuged at 14,000 × g for 15 min at 4 °C. The resulting pellet was collected as the mitochondrial fraction. Protein concentration was determined using Bio-Rad assay. Recombinant Syn was purchased from Chemicon International Inc. (Temecula, CA). Synuclein was diluted in water (to a final concentration of 1 mg/ml), divided into aliquots, and stored at −20 °C until use. In all model experiments in Fig. 1, b–d, and Fig. 2a, the following conditions were utilized: Syn (1.5 μm) was incubated with 0.5 μm cyt c and TOCL/DOPC liposomes (TOCL/Syn ratio 25:1) in 20 mm HEPES, pH 7.4, for 60 min at 37 °C. Incubation volume was 50 μl. 50 μm H2O2 was added to the incubation mixture every 15 min. The reaction was stopped by addition of 5 μl of catalase (0.1 mg/ml).FIGURE 2Formation of hetero-oligomeric complexes and covalent cross-linked aggregates of cyt c/TOCL with wild-type Syn (intact and aged) and its mutants A53T and A30P. a, native PAGE of Syn-cyt c-TOCL hetero-oligomeric complexes (staining with SilverSNAP stain kit, ThermoFisher) (left panel). Syn (8 μm) was incubated with TOCL/DOPC liposomes (TOCL/Syn ratio 20:1) and cyt c (8 μm) for 60 min at 37 °C. Assessment of Syn binding to CL/cyt c using NBD-CL fluorescence (right panel) is shown. Inset, typical fluorescence spectra obtained from the following: CL/NBD-CL liposomes (uppermost curve), CL/NBD-CL-cyt c complexes (lowest curve), and CL/NBD-CL/cyt c after titration with wild-type (Wt) Syn (1) and mutant forms of Syn A53T (2) and A30P (3) (1.75 μm). b, SDS-PAGE (12%) with subsequent Western blot analysis (using anti-Syn antibody) of cross-links formed after incubation of wild-type (wt) Syn, A53T Syn, and A30P Syn with cyt c/TOCL in the presence of H2O2. Wild-type Syn, A30P, and A53T mutants underwent hetero-oligomeric covalent cross-linking with cyt c in a similar way. c, SDS-PAGE (7.5%) with subsequent Western blot analysis (using anti-Syn antibody) of Syn-cyt c-CL covalent cross-links formed by incubation of nonfibrillated and fibrillated (aged) Syn with cyt c/TOCL in the presence of H2O2. Comparable covalent cross-linking of nonfibrillated and fibrillated Syn was observed in the presence of cyt c, TOCL, and H2O2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proteins were separated by SDS-PAGE run in Mini-Protean 3 system (Bio-Rad) in Tris/glycine buffer. 0.8% agarose gel electrophoresis was run in horizontal gel system "Mupid-21" (Cosmo Bio Co., Ltd.) in nondenaturing buffer (43 mm imidazole, 35 mm HEPES, pH 7.4). In experiments with the HeLa cells, proteins were extracted with 1% SDS after cells were washed with PBS. The separated proteins were electrotransferred to nitrocellulose membrane. After blocking with 5% nonfat milk dissolved in phosphate-buffered saline/Tween 20 (PBS-T, 0.05%) or Tris-buffered saline/Tween 20 (TBS-T) for 1 h, membrane was incubated with primary antibodies (anti-synuclein, anti-cyt c, anti-dityrosine, or anti-5,5-dimethyl-1-pyrroline-N-oxide (DMPO) antibodies) overnight at 4 °C. The membranes were washed 3–4 times followed by incubation with horseradish peroxidase- or alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse antibodies for 60 min at room temperature. The protein bands were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce) for horseradish peroxidase-conjugated secondary antibody or Lumi-Phos Western blot (Pierce) for alkaline phosphatase-conjugated secondary antibody as described by the manufacturer. The density of bands was determined by scanning with Epi Chemi II Darkroom (UVP BioImaging Systems, Upland, CA). Peroxidase activity of cyt c was determined by methods utilizing fluorescence, gel electrophoresis, and EPR spectroscopy as follows. (i) Fluorescence of resorufin, an oxidation product of Amplex Red (λex 570 nm, λem 585 nm) was measured using Shimadzu RF-5301PC spectrofluorophotometer (Tokyo, Japan). For determination of peroxidase activity, different amounts of Syn were incubated with 0.5 μm cyt c, liposomes (TOCL/DOPC ratio 1:1; TOCL/cyt c ratio 25:1), 50 μm Amplex Red, 50 mm H2O2. (ii) Peroxidase activity in gel was determined after native electrophoresis in 0.8% agarose. Gels were incubated in solution containing SuperSignal West Pico Chemiluminescent Substrate (Pierce), and chemiluminescence was determined by scanning with Epi Chemi II Darkroom (UVP BioImaging Systems, Upland, CA). (iii) EPR spectroscopy of etoposide phenoxyl radicals produced by oxidation of etoposide was performed at 25 °C under the following conditions: 3,350 G center field; 50 G sweep width, 0.5 G field modulation; 10 milliwatt microwave power; 0.1-s time constant; 2,000 receiver gain; and 4-min time scan. Different amounts of Syn were incubated with 5 μm cyt c, liposomes (TOCL/DOPC ratio 1:1; TOCL/cyt c ratio 25:1), 100 μm etoposide, and 100 μm H2O2. Cyt c (80 μm) was incubated with DOPC/TOCL liposomes (DOPC/TOCL = 1:1; 4 mm total lipid) and Syn for 5 min at room temperature in 25 mm HEPES-Na buffer, pH 7.4, 100 μm DTPA (N2-conditions); then H2O2 (800 μm) was added. The reaction was stopped after 5 s by freezing the samples in liquid nitrogen. The EPR spectra were recorded at 77 K under the following conditions: 3230 G, centered field; 100 G, sweep width; 5 G, field modulation; 5 milliwatts, microwave power; 0.1 s, time constant; and 2-min time scan. Dependence of relative magnitude (percentage of the maximal magnitude) of EPR signals of tyrosyl radicals on square root from microwave power (in milliwatts) was presented as saturation curves. The spin-lattice relaxation time was determined by fitting the experimental curve of radical signal saturation to the theoretical one as described previously (15.Castner T.G.J. Phys. Rev. 1959; 115: 1506-1515Crossref Scopus (240) Google Scholar). Samples were digested with proteinase K (20 μg/ml) at 37 °C overnight. Digested samples were precipitated with cold perchloric acid (final concentration 0.53 n). Samples were allowed to stand 10 min on an ice bath and centrifuged for 15 min at 3,000 × g. After neutralization with 2 n KOH, the precipitate of potassium perchlorate crystals was discarded by centrifugation (15 min at 3,000 × g). Supernatants were diluted with 0.2 m HEPES, pH 9.0, and fluorescence was measured (λex 315, λem 420 nm). CL/cyt c complexes were formed by incubation of DOPC/CL liposomes (5 μm total lipids, including 1 μm CL and 0.05 μm 1,1′,2-trioleoyl-2′-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-cardiolipin (NBD-CL, custom-synthesized by Avanti Polar Lipids, Alabaster, AL) and cyt c (0.05 μm) for 2 min. NBD-CL fluorescence spectra were recorded in the range of 500–650 nm (excitation wavelength of 480 nm, slits 10 and 10 nm) using a Shimadzu F5301-PC spectrofluorometer. NBD fluorescence was monitored 2 min after addition of Syn aliquots (0.25 μm each). After SDS-gel electrophoresis, proteins were visualized by Coomassie staining, and bands corresponding to high molecular weight Syn-cyt c complexes were excised. Destaining was achieved by several washes with 25 mm NH4CO3, 50% CH3CN. Gel pieces were reduced in the presence of dithiothreitol (DTT) followed by alkylation with iodoacetamide. Gel pieces were washed, dehydrated, and dried. The complexes were subjected first to overnight in-gel digestion with trypsin (25 ng/μl in 25 mm ammonium bicarbonate buffer, pH 7.8) followed by overnight digestion with endoproteinase-Glu-C (25 ng/μl in the same buffer). Peptides were extracted with 5% formic acid, 50% acetonitrile and evaporated to near dryness. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a Bruker Ultraflex mass spectrometer in positive reflector mode (20 kV) with a matrix of α-cyano-4-hydroxycinnamic acid (Sigma). At least 500 laser shots were averaged to get each spectrum. Masses were calibrated to known peptide standards (purchased from Applied Biosystems) on the same day of analysis. 30-μl aliquots of each of the digests were acidified with 1.5 μl of 5% trifluoroacetic acid (Sigma), and then taken up into a C18 ZipTip (Millipore) that had been prepared as per the manufacturer's instructions. The bound peptides were desalted with two 15-μl washes of 0.1% trifluoroacetic acid and then eluted with 2 μl of aqueous, acidic acetonitrile (67% CH3CN, 0.1% trifluoroacetic acid). The eluant was mixed with 1 μl of freshly prepared cyano-4-hydroxycinnamic acid stock solution (20 mg/ml cyano-4-hydroxycinnamic acid in aqueous acetonitrile as above), and 1-μl portions of this mixture were spotted onto a MALDI sample plate for air-drying. Potential cross-linked candidates identified by the MALDI run were subjected to sequence analysis by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS). LC-ESI-MS was performed on a Micromass triple quadrupole mass spectrometer (Waters). A microcapillary column (10-cm × 75-μm inner diameter) was packed in-house using 5-μm C18 particles (PerSeptive Biosystems). Flow rates were generated with a Rainin high performance liquid chromatography (HPLC) system equipped with an LC-Packings microflow processor and maintained at 180 nl/min. The Syn-cyt c tryptic/Glu-C digest fragments were loaded onto the microcapillary column, washed in 0.1% acetic acid in water (buffer A), and eluted with a linear gradient of acetonitrile containing 0.1% acetic acid (buffer B) over 30 min. Fragmentation of potential cross-linked species identified by LC-ESI-MS was performed using a collision energy ramp and argon as the collision gas. DOPC/CL liposomes (5 nmol of total lipids, 1:1) were incubated with Syn (100 pmol) for 15 min. The mixture was then incubated in the presence of acridine 10-nonyl bromide (NAO, 2–10 nmol) in 20 mm HEPES buffer, pH 7.4, for 30 min at room temperature. Samples (10 μl) were applied to 7.5% PAGE, and electrophoresis was performed (1 h at 120 V). Unbound Syn was stained with SilverSNAP stain kit from Pierce and quantified by optical density. CL/ Syn binding constants were calculated using Equation 1, {NAO}α-synucleinfree=KLipid-α-synucleinKLipid-NAO(Eq. 1) where Klipid-NAO = 2 × 106 m−1 for CL (16.Petit J.M. Maftah A. Ratinaud M.H. Julien R. Eur. J. Biochem. 1992; 209: 267-273Crossref PubMed Scopus (284) Google Scholar). TLCL and its oxidized species were extracted from the incubation medium by Folch procedure (17.Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and evaporated under N2. DOPC/TLCL liposomes (250 μm at a ratio of 1:1) were incubated with cyt c (5 μm) and H2O2 (100 μm were added every 15 min during incubation) in phosphate-buffered saline (PBS) (pH 7.4 + 100 μm DTPA) in the absence and presence of Syn (15 μm). TLCL hydroperoxides were determined by fluorescence HPLC of products formed in microperoxidase 11-catalyzed reaction with a fluorogenic substrate, Amplex Red, N-acetyl-3,7-dihydroxyphenoxazine (Molecular Probes, Eugene, OR) as described previously (12.Kagan V.E. Tyurin V.A. Jiang J. Tyurina Y.Y. Ritov V.B. Amoscato A.A. Osipov A.N. Belikova N.A. Kapralov A.A. Kini V. Vlasova I.I. Zhao Q. Zou M. Di P. Svistunenko D.A. Kurnikov I.V. Borisenko G.G. Nat. Chem. Biol. 2005; 1: 223-232Crossref PubMed Scopus (951) Google Scholar, 18.Tyurin V.A. Tyurina Y.Y. Kochanek P.M. Hamilton R. DeKosky S.T. Greenberger J.S. Bayır H. Kagan V.E. Methods Enzymol. 2008; 442: 375-393Crossref PubMed Scopus (54) Google Scholar). For ESI-MS analysis, TLCL and its oxidation products were resuspended in chloroform/methanol 1:2 v/v (20 pmol/μl) and analyzed by direct infusion (flow rate of 5 μl/min) into a quadrupole linear ion trap mass spectrometer LXQ (Thermo Electron, San Jose, CA) The electrospray probe was operated at a voltage differential of 5 kV in the negative ion mode. Source temperature was maintained at 150 °C. Human HL-60 cells were grown in RPMI 1640 medium supplemented with 15% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. The cytosol extracts (S-100) were obtained as described previously (19.Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar) with minor modification. Briefly, the cells were washed twice in cold phosphate-buffered saline, pH 7.4, and the resulting pellet was resuspended in buffer containing 25 mm HEPES-KOH, pH 7.0, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride, 0.05% digitonin, and 1% protease inhibitor mixture (Sigma) for 2 min at 4 °C. Cells were centrifuged at 4 °C for 10 min at 10,000 × g. The resulting supernatant was further centrifuged at 4 °C for 50 min at 100,000 × g. The supernatant was collected as S-100 and kept at −80 °C until further use. For caspase-3 activation, S-100 (5 μg/μl) was incubated with 1 mm dATP and 1 μm cyt c for 90 min at 37 °C, and caspase-3 activity was normalized as 100%. Syn (13 μm) was added alone or in complex with TOCL (at a ratio of 3:1). The caspase-3 activity was evaluated as described in the manufacturer's manual (Invitrogen, Enzchek caspase-3 assay kit). All animal use was in accordance with National Institutes of Health guidelines and was approved by the Pittsburgh University Institutional Animal Care and Use Committee. Surgeries were performed as described previously (20.Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2863) Google Scholar). Briefly, male Lewis rats (300–350 g) received 3.0 mg/kg/day rotenone for up to 4 weeks through subcutaneous osmotic mini-pumps (Alzet Corp., Palo Alto, CA). Control rats received vehicle (DMSO/polyethylene glycol, 1:1). Rotenone-infused rats were euthanized at the time of severe systemic illness characterized by rigidity and akinesia that prevented adequate feeding and grooming. Control rats were eutha