Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H2O2 resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD. Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H2O2 resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD. Parkinson's disease (PD) 1The abbreviations used are:PDParkinson's diseaseaSynα-synucleinROSreactive oxygen speciesIPTGisopropylthiogalactosideTCEPtris(2-carboxyethyl) phosphine2D-PAGEtwo-dimensional polyacrylamide gel electrophoresisBCAbicinchoninic acidfar-UV CDfar-ultraviolet circular dichroismIPGimmobilized pH gradientGSTglutathione-S-transferase. 1The abbreviations used are:PDParkinson's diseaseaSynα-synucleinROSreactive oxygen speciesIPTGisopropylthiogalactosideTCEPtris(2-carboxyethyl) phosphine2D-PAGEtwo-dimensional polyacrylamide gel electrophoresisBCAbicinchoninic acidfar-UV CDfar-ultraviolet circular dichroismIPGimmobilized pH gradientGSTglutathione-S-transferase. is a neurodegenerative disorder characterized by muscular rigidity, slowness of voluntary movement, poor balance, and resting tremor (1Dawson T.M. Dawson V.L. Molecular pathways of neurodegeneration in Parkinson's disease.Science. 2003; 302: 819-822Crossref PubMed Scopus (1393) Google Scholar). These symptoms are caused by the death of neurons in a region of the midbrain called the substantia nigra. The neurons that survive in this region exhibit a defect in complex I of the mitochondrial electron transport chain and show signs of oxidative damage (2Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease.Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2933) Google Scholar, 3Banerjee R. Starkov A.A. Beal M.F. Thomas B. Mitochondrial dysfunction in the limelight of Parkinson's disease pathogenesis.Biochim. Biophys. Acta. 2009; 1792: 651-663Crossref PubMed Scopus (197) Google Scholar, 4Malkus K.A. Tsika E. Ischiropoulos H. Oxidative modifications, mitochondrial dysfunction, and impaired protein degradation in Parkinson's disease: how neurons are lost in the Bermuda triangle.Mol. Neurodegener. 2009; 4: 24Crossref PubMed Scopus (95) Google Scholar). In addition, surviving neurons contain characteristic cytosolic inclusions named "Lewy bodies" that are enriched with aggregated forms of the presynaptic protein α-synuclein (aSyn) (5Spillantini M.G. Schmidt M.L. Lee V. M.-Y. Trojanowski J.Q. Jakes R. Goedert M. α-Synuclein in Lewy bodies.Nature. 1997; 388: 839-840Crossref PubMed Scopus (6103) Google Scholar). It is hypothesized that reactive oxygen species (ROS) accumulate as a result of mitochondrial impairment and contribute to neurodegeneration by causing the oxidation of lipid, proteins, and DNA (2Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease.Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2933) Google Scholar, 3Banerjee R. Starkov A.A. Beal M.F. Thomas B. Mitochondrial dysfunction in the limelight of Parkinson's disease pathogenesis.Biochim. Biophys. 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ROS accumulation is thought to occur preferentially in nigral dopaminergic neurons because of the catabolism and auto-oxidation of dopamine, reactions that result in the generation of H2O2 (2Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease.Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2933) Google Scholar, 9Graham D.G. Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones.Mol. Pharmacol. 1978; 14: 633-643PubMed Google Scholar). In addition, an abundance of iron in the substantia nigra promotes the decomposition of H2O2 to OH· by Fenton chemistry (10Hirsch E.C. Faucheux B.A. Iron metabolism and Parkinson's disease.Mov. Disord. 1998; 13: 39-45PubMed Google Scholar). Parkinson's disease α-synuclein reactive oxygen species isopropylthiogalactoside tris(2-carboxyethyl) phosphine two-dimensional polyacrylamide gel electrophoresis bicinchoninic acid far-ultraviolet circular dichroism immobilized pH gradient glutathione-S-transferase. Parkinson's disease α-synuclein reactive oxygen species isopropylthiogalactoside tris(2-carboxyethyl) phosphine two-dimensional polyacrylamide gel electrophoresis bicinchoninic acid far-ultraviolet circular dichroism immobilized pH gradient glutathione-S-transferase. A number of patients with familial, early-onset, recessive PD have been found to harbor loss-of-function mutations (e.g. M26I, E64D, A104T, D149A, E163K, and L166P) in the gene encoding DJ-1, a protein that is abundant throughout the central nervous system (11Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. 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Yang L. Beal M.F. Nishimura I. Wakamatsu K. Ito S. Takahashi R. Lu B. Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13670-13675Crossref PubMed Scopus (306) Google Scholar, 25Aleyasin H. Rousseaux M.W. Marcogliese P.C. Hewitt S.J. Irrcher I. Joselin A.P. Parsanejad M. Kim R.H. Rizzu P. Callaghan S.M. Slack R.S. Mak T.W. Park D.S. DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3186-3191Crossref PubMed Scopus (123) Google Scholar). DJ-1 is a homodimer (subunit molecular weight = 20 kDa) for which there is evidence that cysteine 106 (C106) in the subunit sequence is readily oxidized to the sulfinic acid under oxidizing conditions, yielding the "2O" form of the protein (14Meulener M.C. Xu K. Thomson L. Thompson L. Ischiropoulos H. Bonini N.M. 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Acad. Sci. U.S.A. 2007; 104: 14807-14812Crossref PubMed Scopus (388) Google Scholar). Structural studies indicate that C106 is located in a pocket at the interface between the DJ-1 subunits that is lined with polar residues from both subunits, and oxidation of C106 to the sulfinic acid is facilitated by structure(s) within the pocket (16Canet-Avilés R.M. Wilson M.A. Miller D.W. Ahmad R. McLendon C. Bandyopadhyay S. Baptista M.J. Ringe D. Petsko G.A. Cookson M.R. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9103-9108Crossref PubMed Scopus (908) Google Scholar, 27Tao X. Tong L. Crystal structure of human DJ-1, a protein associated with early onset Parkinson's disease.J. Biol. Chem. 2003; 278: 31372-31379Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 28Wilson M.A. Collins J.L. Hod Y. Ringe D. Petsko G.A. 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U.S.A. 2004; 101: 9103-9108Crossref PubMed Scopus (908) Google Scholar), inhibit mitochondrial fragmentation (30Blackinton J. Lakshminarasimhan M. Thomas K.J. Ahmad R. Greggio E. Raza A.S. Cookson M.R. Wilson M.A. Formation of a stabilized cysteine sulfinic acid is critical for the mitochondrial function of the parkinsonism protein DJ-1.J. Biol. Chem. 2009; 284: 6476-6485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 31Irrcher I. Aleyasin H. Seifert E.L. Hewitt S.J. Chhabra S. Phillips M. Lutz A.K. Rousseaux M.W. Bevilacqua L. Jahani-Asl A. Callaghan S. MacLaurin J.G. Winklhofer K.F. Rizzu P. Rippstein P. Kim R.H. Chen C.X. Fon E.A. Slack R.S. Harper M.E. McBride H.M. Mak T.W. Park D.S. Loss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics.Hum. Mol. Genet. 2010; 19: 3734-3746Crossref PubMed Scopus (295) Google Scholar, 32Kamp F. Exner N. Lutz A.K. Wender N. Hegermann J. Brunner B. Nuscher B. Bartels T. Giese A. Beyer K. Eimer S. Winklhofer K.F. Haass C. Inhibition of mitochondrial fusion by alpha-synuclein is rescued by PINK1, Parkin and DJ-1.EMBO J. 2010; 29: 3571-3589Crossref PubMed Scopus (376) Google Scholar), or suppress fibril formation by recombinant aSyn (24Zhou W. Zhu M. Wilson M.A. Petsko G.A. Fink A.L. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein.J. Mol. Biol. 2006; 356: 1036-1048Crossref PubMed Scopus (305) Google Scholar). In contrast, overoxidation of DJ-1 leading to the conversion of C106 to the sulfonic acid ("3O") form results in thermodynamic instability (15Hulleman J.D. Mirzaei H. Guigard E. Taylor K.L. Ray S.S. Kay C.M. Regnier F.E. Rochet J.C. Destabilization of DJ-1 by familial substitution and oxidative modifications: implications for Parkinson's disease.Biochemistry. 2007; 46: 5776-5789Crossref PubMed Scopus (44) Google Scholar) and a loss of chaperone function (24Zhou W. Zhu M. Wilson M.A. Petsko G.A. Fink A.L. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein.J. Mol. Biol. 2006; 356: 1036-1048Crossref PubMed Scopus (305) Google Scholar). Data from various groups indicate that L166P has a pronounced protein folding defect, resulting in impaired homodimer formation, rapid protein turnover, and a high propensity to form large protein complexes (33Macedo M.G. Anar B. Bronner I.F. Cannella M. Squitieri F. Bonifati V. Hoogeveen A. Heutink P. Rizzu P. The DJ-1 L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes.Hum. Mol. Genet. 2003; 12: 2807-2816Crossref PubMed Scopus (126) Google Scholar, 34Moore D.J. Zhang L. Dawson T.M. Dawson V.L. A missense mutation (L166P) in DJ-1, linked to familial Parkinson's disease, confers reduced protein stability and impairs homo-oligomerization.J. Neurochem. 2003; 87: 1558-1567Crossref PubMed Scopus (189) Google Scholar, 35Olzmann J.A. Brown K. Wilkinson K.D. Rees H.D. Huai Q. Ke H. Levey A.I. Li L. Chin L.S. Familial Parkinson's disease-associated L166P mutation disrupts DJ-1 protein folding and function.J. Biol. Chem. 2004; 279: 8506-8515Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Clearly, pronounced structural defects lead to the compromised functionality of L166P. Substitutions at other locations destabilize the structure of DJ-1 to a lesser extent (15Hulleman J.D. Mirzaei H. Guigard E. Taylor K.L. Ray S.S. Kay C.M. Regnier F.E. Rochet J.C. Destabilization of DJ-1 by familial substitution and oxidative modifications: implications for Parkinson's disease.Biochemistry. 2007; 46: 5776-5789Crossref PubMed Scopus (44) Google Scholar, 36Lakshminarasimhan M. Maldonado M.T. Zhou W. Fink A.L. Wilson M.A. Structural impact of three Parkinsonism-associated missense mutations on human DJ-1.Biochemistry. 2008; 47: 1381-1392Crossref PubMed Scopus (39) Google Scholar, 37Malgieri G. Eliezer D. Structural effects of Parkinson's disease linked DJ-1 mutations.Protein Sci. 2008; 17: 855-868Crossref PubMed Scopus (58) Google Scholar), raising questions about why they are functionally diminished. As one possibility, we hypothesized that some familial DJ-1 mutants may have altered abilities to undergo key oxidative modifications, namely, (1) a decreased propensity to undergo oxidation at position 106 to yield the 2O form, and/or (2) an increased susceptibility to undergo oxidative modifications with potentially deleterious effects on DJ-1 function at other sites in the polypeptide chain. To address this hypothesis, we examined the impact of one familial substitution (the M26I mutation) on the ability of DJ-1 to undergo oxidation at C106 and other residues in the amino acid sequence. Using a novel mass spectrometry approach, we quantified site-specific oxidative modifications of M26I as compared with wild-type DJ-1 after treatment with different amounts of H2O2. Our results indicate that the M26I substitution has a profound disruptive effect on the ability of DJ-1 to undergo oxidation at C106. Unless otherwise specified, chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Isopropylthiogalactoside (IPTG) was purchased from Gold Biotechnologies (St. Louis, MO). PreScission™ Protease was obtained from GE Healthcare (Piscataway, NJ). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Biotechnology (Rockford, IL). Immobilized trypsin and tris (2-carboxyethyl) phosphine (TCEP) were purchased as Pierce products from Thermofisher Scientific (Rockford, IL). Amicon Ultra-0.5 ml (Ultracel YM-10) and Amicon Ultra-4 (Ultracel-3) centrifugal filters and H2O2 were purchased from Millipore (Billerica, MA). Trifluoroacetic acid and high-performance liquid chromatography (HPLC) grade CH3CN were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Materials for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (immobilized pH gradient (IPG) strips, sample rehydration buffer, Criterion XT 12% Bis-Tris precast gels, XT MOPS 2D PAGE running buffer, protein standards) were obtained from Bio-Rad (Hercules, CA). Urea was obtained from Mallinckrodt Laboratories (Phillipsburg, NJ), and agarose was purchased from Invitrogen (Carlsbad, CA). Human wild-type DJ-1 and the familial mutant M26I were expressed as N-terminal glutathione-S-transferase (GST) fusions. A construct encoding GST-DJ-1M26I in the pGEX-6P1 vector (courtesy of Dr. Soumya Ray, Brigham and Women's Hospital (38Logan T. Clark L. Ray S.S. Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease.Biochemistry. 2010; 49: 5624-5633Crossref PubMed Scopus (8) Google Scholar)) was converted to a new construct encoding GST-DJ-1WT by PCR using the QuikChange method (Stratagene, La Jolla, CA). The sequence of the DJ-1-encoding insert in each construct was verified using an Applied Biosystems (Foster City, CA; ABI 3700) DNA sequencer. Wild-type and mutant DJ-1 were purified as described (38Logan T. Clark L. Ray S.S. Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease.Biochemistry. 2010; 49: 5624-5633Crossref PubMed Scopus (8) Google Scholar). Cells of the BL21(DE3) strain of Escherichia coli were transformed with each pGEX-6P-1 GST-DJ-1 construct by electroporation. The transformed cells were grown to an OD600 of 0.4–0.6 in LB plus ampicillin (100 μg/ml) at 37 °C, and IPTG was added to a final concentration of 1 mm. The cells were grown under inducing conditions for 18 h at 18 °C, harvested by centrifugation, and resuspended in buffer G (25 mm KPi, pH 7.0, 200 mm KCl). The cells were lysed by incubation on ice in the presence of lysozyme (1 mg/ml, 30 min) followed by passage through a French pressure cell (p.s.i. > 1000). After centrifugation (20,000 × g, 20 min), the supernatant was applied to a GSTPrep FF column (GE Healthcare), from which GST-DJ-1 was eluted in 250 mm Tris HCl, pH 8.0, 500 mm NaCl, and 0.3% [w/v] reduced glutathione. Fractions most highly enriched with GST-DJ-1 were identified by SDS-PAGE with Coomassie Blue staining and pooled. The pooled fractions were dialyzed against buffer G plus dithiothreitol (DTT) (0.25 mm) to remove excess reduced glutathione. The overall protein concentration was determined with a BCA protein assay kit, and the fusion protein was cleaved with PreScission™ Protease (16 h, 4 °C, 1 unit protease per 133 μg DJ-1). Untagged DJ-1 was separated from uncleaved GST-DJ-1, free GST, and residual protease (which contains an uncleavable GST tag) by elution from a GSTPrep FF column equilibrated with buffer G. Fractions most highly enriched with DJ-1 were identified by SDS-PAGE with Coomassie Blue staining and pooled. The final protein sample (estimated purity, 95%) was supplemented with glycerol (5%, [v/v]) and DTT (2–3 mm), and aliquots were frozen at −80 °C. For all of the analyses outlined below, the concentration of recombinant DJ-1 was estimated using the BCA assay and verified with quantitative amino acid analysis (Purdue University Proteomics Core). Aliquots of purified DJ-1 (wild-type or M26I) were dialyzed against 10 mm Tris at pH 8.0 to remove reductant, and the protein was treated with a 10-fold or 500-fold molar excess of H2O2 for 1 h at 22 °C. These conditions were previously found to be suitable to convert DJ-1 to the 2O or 3O form, respectively (24Zhou W. Zhu M. Wilson M.A. Petsko G.A. Fink A.L. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein.J. Mol. Biol. 2006; 356: 1036-1048Crossref PubMed Scopus (305) Google Scholar). Excess H2O2 was removed from each sample by exchanging the protein into fresh buffer (10 mm Tris at pH 8.0) using Amicon Ultra-4 centrifugal filters (molecular weight cutoff, 3 kDa). Changes in the isoelectric point (pI) of DJ-1 following oxidation in the presence of H2O2 as outlined above were monitored via 2D-PAGE (16Canet-Avilés R.M. Wilson M.A. Miller D.W. Ahmad R. McLendon C. Bandyopadhyay S. Baptista M.J. Ringe D. Petsko G.A. Cookson M.R. The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9103-9108Crossref PubMed Scopus (908) Google Scholar). Protein aliquots were dialyzed overnight against PBS. An aliquot of the protein (15 μg) was mixed with sample rehydration buffer (8 m urea, 2% [w/v] 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 50 mm DTT, 0.2% [w/v] BioLyte 3/10 ampholyte, 0.001% [w/v] bromphenol blue) and recombinant aSyn as an internal standard (predicted pI = 4.67) and Bio-Rad protein standards in a total volume of 185 μl. The solution was added to an 11 cm IPG strip with a pH range of 4 to 7. Mineral oil was added to the top of the IPG strip to reduce evaporation during electrophoresis. The IPG strip was actively rehydrated at 20 °C for 12 h and subjected to isoelectric focusing in a Protean isoelectric focusing (IEF) Cell (Bio-Rad) with the following voltage parameters: step one, 250 V for 15 min; step two, 8000 V for 2.5 h; step 3, 8000 V for 4.4 h. The IPG strip was reduced with 2% [w/v] DTT in equilibration buffer (6 m urea, 0.375 m Tris HCl, pH 8.8, 2% [w/v] SDS, 20% [v/v] glycerol) for 15 min, followed by alkylation with 2.5% [w/v] iodoacetamide for 15 min. The IPG strip was loaded onto the top of a Criterion XT 12% Bis-Tris pre-cast gel (Bio-Rad) and sealed with 0.5% [w/v] agarose in 1 × XT MOPS buffer to ensure an even transfer of protein. The second-dimension gel was stained with Coomassie Blue and analyzed with a Typhoon Imaging System. Approximate pI values were determined by calibration with the aSyn internal standard and the ends of the IPG strip. Intensities of spots on 2D-PAGE gels were quantitated using Image J software (National Institutes of Health). Each spot was selected using a rectangular selection tool. The dimensions of the selected rectangular area were kept constant through the analysis of all spots on multiple 2D-PAGE gels. Each selected spot was displayed as a peak, to which a baseline was manually fitted, and the area under the peak was calculated. After correcting for background (determined from a rectangular zone in an "empty" region of the 2D-PAGE gel), peak areas were used to calculate the relative amount of 2O isoform in each samp