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• W2063089997 abstract "The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1. The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1. Reactive cysteine residues are susceptible to a variety of covalent modifications that are increasingly recognized as a major means of regulating the activities of many proteins (1Reddie K.G. Carroll K.S. Curr. Opin. Chem. Biol. 2008; 12: 746-754Crossref PubMed Scopus (508) Google Scholar). Cysteine forms three different species by the direct addition of oxygen; cysteine-sulfenic (-SOH), -sulfinic (-SO2H), and -sulfonic (-SO3H) acid. Because cysteine can be oxidized to three distinct species, each with different structural and chemical properties, cysteine oxidation is a versatile way for reactive oxygen species (ROS) 4The abbreviations used are: ROS, reactive oxygen species; ANOVA, analysis of variance; DTT, dithiothreitol; FRAP, fluorescence recovery after photobleaching; LC-MS/MS, liquid chromatography-mass spectrometry; MEF, mouse embryonic fibroblast; VDAC1, voltage-dependent anion channel 1; WT, wild type. to alter the activity of a protein. Of the three oxidation products of cysteine, only cysteine-sulfenic acid is readily reduced to the thiol under physiological conditions. However, enzymes that catalyze the ATP-dependent reduction of overoxidized peroxiredoxins containing cysteine-sulfinic acid to cysteine have been discovered and characterized (2Biteau B. Labarre J. Toledano M.B. Nature. 2003; 425: 980-984Crossref PubMed Scopus (788) Google Scholar, 3Jonsson T.J. Murray M.S. Johnson L.C. Poole L.B. Lowther W.T. Biochemistry. 2005; 44: 8634-8642Crossref PubMed Scopus (48) Google Scholar). With reversibility comes the potential for cysteine-sulfinic acid modifications to modulate the function of various target proteins in a redox-dependent manner. Therefore, at least in some proteins, cysteine-sulfinic acid should be regarded as a post-translational modification rather than simply a type of protein damage.As expected, many of the proteins that are modified by cysteine oxidation are involved in the oxidative stress response or in the maintenance of cellular redox homeostasis. Of these proteins, DJ-1 has special importance in understanding the role of regulatory cysteine oxidation in neuronal survival. Loss of function mutations in DJ-1 are a rare cause of early onset recessive parkinsonism (4Annesi G. Savettieri G. Pugliese P. D’Amelio M. Tarantino P. Ragonese P. La Bella V. Piccoli T. Civitelli D. Annesi F. Fierro B. Piccoli F. Arabia G. Caracciolo M. Cirò Candiano I.C. Quattrone A. Ann. Neurol. 2005; 58: 803-807Crossref PubMed Scopus (135) Google Scholar, 5Bonifati 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. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2195) Google Scholar), although the exact function of DJ-1 is unclear. The protein is part of the large DJ-1 superfamily with evolutionarily conserved members in bacteria, fungi, plants, and animals (6Bandyopadhyay S. Cookson M.R. BMC Evol. Biol. 2004; 4: 6Crossref PubMed Scopus (128) Google Scholar, 7Lucas J.I. Marin I. Mol. Biol. Evol. 2007; 24: 551-561Crossref PubMed Scopus (56) Google Scholar). A number of activities have been proposed for human DJ-1, including a weak peroxiredoxin-like activity (8Andres-Mateos E. Perier C. Zhang L. Blanchard-Fillion B. Greco T.M. Thomas B. Ko H.S. Sasaki M. Ischiropoulos H. Przedborski S. Dawson T.M. Dawson V.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14807-14812Crossref PubMed Scopus (385) Google Scholar), a chaperone activity (9Shendelman S. Jonason A. Martinat C. Leete T. Abeliovich A. PLoS Biol. 2004; 2: 1764-1773Crossref Scopus (490) Google Scholar, 10Zhou W. Zhu M. Wilson M.A. Petsko G.A. Fink A.L. J. Mol. Biol. 2006; 356: 1036-1048Crossref PubMed Scopus (304) Google Scholar), and translational (11Hod Y. Pentyala S.N. Whyard T.C. El-Maghrabi M.R. J. Cell Biochem. 1999; 72: 435-444Crossref PubMed Scopus (170) Google Scholar, 12van der Brug M.P. Blackinton J. Chandran J. Hao L.Y. Lal A. Mazan-Mamczarz K. Martindale J. Xie C. Ahmad R. Thomas K.J. Beilina A. Gibbs J.R. Ding J. Myers A.J. Zhan M. Cai H. Bonini N.M. Gorospe M. Cookson M.R. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10244-10249Crossref PubMed Scopus (175) Google Scholar) and transcriptional regulation (13Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Zhou W. Freed C.R. J. Biol. Chem. 2005; 280: 43150-43158Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar).The best-established aspect of DJ-1 function is its ability to respond to oxidative stress. DJ-1 is modified under oxidative stress both in vitro and in vivo by oxidation of a very highly conserved cysteine residue (Cys106 in human DJ-1) to form a cysteine-sulfinic acid (Cys106-SO2-) (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar). Several studies have shown that of the three cysteine residues in human DJ-1, Cys106 is the most prone to oxidative modification (8Andres-Mateos E. Perier C. Zhang L. Blanchard-Fillion B. Greco T.M. Thomas B. Ko H.S. Sasaki M. Ischiropoulos H. Przedborski S. Dawson T.M. Dawson V.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14807-14812Crossref PubMed Scopus (385) Google Scholar, 16Ito G. Ariga H. Nakagawa Y. Iwatsubo T. Biochem. Biophys. Res. Commun. 2006; 339: 667-672Crossref PubMed Scopus (60) Google Scholar, 17Kinumi T. Kimata J. Taira T. Ariga H. Niki E. Biochem. Biophys. Res. Commun. 2004; 317: 722-728Crossref PubMed Scopus (299) Google Scholar). In addition, Cys106 has a low pKa value of 5.4 and therefore exists almost exclusively as the highly reactive cysteine thiolate anion at physiological pH (18Witt A.C. Lakshminarasimhan M. Remington B.C. Hasim S. Pozharski E. Wilson M.A. Biochemistry. 2008; 47: 7430-7440Crossref PubMed Scopus (91) Google Scholar). Replacement of Cys106 with other amino acids in DJ-1 results in a loss of protective activity against oxidative stressors in a number of systems (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar, 19Aleyasin H. Rousseaux M.W. Phillips M. Kim R.H. Bland R.J. Callaghan S. Slack R.S. During M.J. Mak T.W. Park D.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18748-18753Crossref PubMed Scopus (137) Google Scholar, 20Meulener M. Whitworth A.J. Armstrong-Gold C.E. Rizzu P. Heutink P. Wes P.D. Pallanck L.J. Bonini N.M. Curr. Biol. 2005; 15: 1572-1577Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar).We have therefore previously suggested that formation of cysteine-sulfinic acid is required for DJ-1 to exert its protective effects (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar). However, the substitution of the equivalent cysteine residue in Drosophila melanogaster DJ-1 with aspartic acid (C104D in Drosophila) inactivates DJ-1, suggesting that the simple addition of a negatively charged residue at this position is insufficient to support DJ-1 function (21Meulener M.C. Xu K. Thomson L. Thompson L. Ischiropoulos H. Bonini N.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12517-12522Crossref PubMed Scopus (193) Google Scholar). As a consequence of the need for direct mutation of Cys106 in these studies, it is unclear whether the cysteine residue itself or its oxidation to a sulfinic acid is critical for the protective activity of DJ-1.We have previously shown using x-ray crystallography that Cys106-SO2- interacts with nearby residues, most notably the highly conserved Glu18 residue (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar). In reduced DJ-1, the carboxylic acid side chain of Glu18 is protonated and donates a hydrogen bond to Cys106, which depresses the thiol pKa value (18Witt A.C. Lakshminarasimhan M. Remington B.C. Hasim S. Pozharski E. Wilson M.A. Biochemistry. 2008; 47: 7430-7440Crossref PubMed Scopus (91) Google Scholar). Therefore, we hypothesized that modifying the environment around the side chain of Cys106 could decouple the oxidation propensity and pKa of Cys106 without changing the cysteine residue itself. In the present study, we have tested this approach by characterizing the effect of Glu18 mutations on the oxidative propensity and cytoprotective activity of Cys106. Our results show that the formation of Cys106-SO2- is critical for DJ-1 to protect cells against mitochondrial damage. In addition, this targeted mutagenesis strategy could be used to manipulate the oxidation state of other cysteine redox-regulated proteins of known structure.EXPERIMENTAL PROCEDURESProtein Expression and Purification-Wild-type and mutant DJ-1 variants were cloned between the NdeI and XhoI sites of the bacterial expression vector pET21a and expressed in BL21(DE3) Escherichia coli (Novagen). All proteins were expressed with a noncleavable C-terminal His6 tag (vector-derived sequence LEHHHHHH) for purification by metal affinity Ni2+-nitrilotriacetic acid chromatography. Bacteria were grown in LB medium supplemented with 100 μg/ml ampicillin at 37 °C with shaking. Once the A600 of the culture reached 0.5-0.7, it was equilibrated at 20 °C for 3 h prior to induction of protein expression by the addition of 0.1 mm isopropyl β-d-1-thiogalactopyranoside. The induced culture was incubated at 20 °C with shaking overnight and harvested by centrifugation. Cell pellets were stored at -80 °C until needed.Recombinant His6-tagged proteins were purified using Ni2+-nitrilotriacetic acid His-Select resin (Sigma). Eluted DJ-1 protein was dialyzed against storage buffer (25 mm HEPES, pH 7.5, 100 mm KCl, 1 mm dithiothreitol (DTT)), loaded onto an equilibrated High Q anion exchange column, and collected in the flow-through, since contaminants bind to the anion exchange resin under these conditions. Purified DJ-1 was concentrated to 1 mm (∈280 = 4000 m-1 cm-1) and ran as a single band on overloaded Coomassie-stained SDS-PAGE. The purified protein was supplemented with 2 mm DTT, snap-frozen on liquid nitrogen, and stored at -80 °C.Hydrogen Peroxide Titration of DJ-1-The in vitro oxidative susceptibility of Cys106 in DJ-1 was assayed by titration with several molar ratios of H2O2 to protein monomer. Thawed DJ-1 was rapidly exchanged into extensively degassed nanopure water using a centrifugal spin column containing P6-DG desalting resin (Bio-Rad). Control experiments using degassed buffered solutions (10 mm potassium phosphate, pH 7.4) instead of water showed similar oxidative behavior of DJ-1 but gave noisier mass spectrometry data due to the presence of buffer salts. Freshly diluted H2O2 was added to DJ-1 in molar ratios of 0:1, 0.5:1, 1:1, 2.5:1, 5:1, 7.5:1, and 10:1 H2O2/protein monomer and incubated on ice for 30 min. Excess H2O2 was removed using a P6-DG centrifugal desalting column, and the protein samples were immediately supplemented with 5 mm DTT, frozen on liquid nitrogen, and stored at -80 °C. DTT, which cannot reduce cysteine-sulfinic acid, was added to ensure that the sample did not further oxidize during sample handling for mass spectrometric analysis. Previous results have indicated that only Cys106 is oxidized by these conditions in vitro (10Zhou W. Zhu M. Wilson M.A. Petsko G.A. Fink A.L. J. Mol. Biol. 2006; 356: 1036-1048Crossref PubMed Scopus (304) Google Scholar).Mass Spectrometry of Oxidized DJ-1-Intact DJ-1 protein was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) with a 4000 Q-trap mass spectrometer (ABS) using a turbo ion spray source probe at the University of Nebraska Redox Biology Center Mass Spectrometry Core Facility. Protein samples (20 μl) were loaded onto a C18 reverse phase column using a PE 200 Autosampler. A SCL-10A high performance liquid chromatography system (Shimadzu) was used for room temperature gradient elution at a flow rate of 100 ml/min in 5 min by using a linear gradient from 0.3% formic acid in water (Solvent A) to 0.3% formic acid in acetonitrile (Solvent B). The elution time for DJ-1 was about 4 min. Data were acquired and processed using Analyst 1.4.1 software in Q1 (quadrupole one)-positive ion mode, and the m/z range of 880-1120 atomic mass units was scanned in 4 s. The total run time for each sample was 10 min. The molecular mass of protein was generated from several multiply charged peaks using the Bayesian Protein Reconstruct option in BioAnalyst 1.4 software. For all experiments, only two species were observed: the reduced protein and an adduct at +32 atomic mass units, corresponding to the Cys106-SO2- form of DJ-1.Crystallization, Data Collection, and Processing-For all crystallization experiments, DJ-1 at 1 mm (20 mg/ml) in storage buffer was crystallized using the hanging drop vapor diffusion method with drops containing 2 μl of protein and 2 μl of reservoir solution. Crystals of E18Q DJ-1 in space group P3121 were grown in 2-5 days at room temperature using a reservoir solution of 30% polyethylene glycol 400, 50 mm HEPES, pH 7.5, 125 mm sodium citrate. Crystals of E18D DJ-1 in space group P3121 were grown from 1.3-1.5 m sodium citrate, 50 mm HEPES, pH 7.5. For crystals of E18Q DJ-1, the 30% polyethylene glycol 400 in the mother liquor was sufficient for cryoprotection. E18D DJ-1 crystals were cryoprotected in 2.4 m sodium malonate, pH 7.0 (22Holyoak T. Fenn T.D. Wilson M.A. Moulin A.G. Ringe D. Petsko G.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2356-2358Crossref PubMed Scopus (47) Google Scholar). All crystals were transferred to nylon loops and cryocooled by direct immersion into liquid nitrogen.Diffraction data were collected at the Advanced Photon Source, BioCARS beamline 14BM-C using 13.776 keV (0.9 Å) incident x-rays and an ADSC Q315 detector. Single crystals maintained at 100 K were used for the collection of each data set, and both data sets were collected in separate high and a low resolution passes with differing exposure times, oscillation ranges, and detector distances to record the full dynamic range of the diffraction data. To avoid radiation-induced damage to Cys106, the x-ray beam was attenuated, and the crystal was exposed to x-rays for 5 s or less per 1° oscillation. Diffraction data were integrated and scaled using HKL2000 (23Otwinowski Z. Minor W. Methods Enzymol. 1997; : 307-326Crossref Scopus (38361) Google Scholar), and final data statistics for each data set are provided in Table 1.TABLE 1Data collection and refinement statisticsOxidized E18D DJ-1Oxidized E18Q DJ-1Data collectionSpace groupP3121P3121Cell dimensionsa = b, c (Å)74.75, 74.8074.77, 74.79Resolution (Å)aValues in parentheses are for the highest resolution shell.30-1.20 (1.24-1.20)30-1.15 (1.18-1.15)RmergebRmerge = , where i is the ith observation of a reflection with indices, h, k, and l, and angle brackets indicate the average over all i observations.0.066 (0.640)0.092 (0.379)<I>/<σ(I)>32.0 (3.7)25.7 (6.9)Completeness (%)99.8 (100)99.8 (100)Redundancy9.9 (9.5)10.0 (9.8)RefinementResolution (Å)30-1.2030-1.15No. of reflections75,73185,756Rwork; Rwork for Fo > 4σ(Fo) (%)cRwork = , whereFhklc is the calculated structure factor amplitude with indices h, k, and l.12.2; 11.011.6; 10.0Rfree; Rfree for Fo > 4σ(Fo) (%)dRfree is calculated as Rwork, where theFhklc values are taken from a test set comprising 5% of the data that were excluded from the refinement (25).15.5; 14.214.5; 13.6Rall; Rall for Fo > 4σ(Fo) (%)eRall is calculated as Rwork, where theFhklc values include all measured data (including the Rfree test set).12.4; 11.111.6; 10.0No. of atomsProtein14581439Water261288Beq factors (Å2)Protein15.016.6Water34.932.6Root mean square deviationsBond lengths (Å)0.0140.015Bond angle 1-3 distances (Å)0.0300.030a Values in parentheses are for the highest resolution shell.b Rmerge = , where i is the ith observation of a reflection with indices, h, k, and l, and angle brackets indicate the average over all i observations.c Rwork = , whereFhklc is the calculated structure factor amplitude with indices h, k, and l.d Rfree is calculated as Rwork, where theFhklc values are taken from a test set comprising 5% of the data that were excluded from the refinement (25Brunger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3849) Google Scholar).e Rall is calculated as Rwork, where theFhklc values include all measured data (including the Rfree test set). Open table in a new tab Crystal Structure Refinement-SHELX-97 was used for refinement of coordinates and atomic displacement parameters against a least squares intensity-based residual target function (24Sheldrick G.M. Acta Crystallogr. A. 2008; 64: 112-122Crossref PubMed Scopus (79888) Google Scholar). All refinements excluded a test set of 5% of randomly chosen reflections that were sequestered and used for the calculation of the Rfree value (25Brunger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3849) Google Scholar). For both structures, initial rigid body refinement at 2.5 Å resolution using coordinates for human DJ-1 (Protein Data Bank code 1P5F) (26Wilson M.A. Collins J.L. Hod Y. Ringe D. Petsko G.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9256-9261Crossref PubMed Scopus (254) Google Scholar) was followed by multiple cycles of restrained refinement of coordinates and isotropic B-factors at 1.5 Å resolution using a stepwise increase in resolution (STIR instruction). Manual adjustments to the model were made by inspection of 2mFo - DFc and mFo - DFc electron density maps using the program COOT (27Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar). Additional cycles of conjugate gradient refinement were performed after inclusion of all data to the full resolution limit of each data set. In the later stages of refinement, anisotropic atomic displacement parameters were introduced, resulting in a 3-5% decrease in both R and Rfree. The final cycles of refinement were performed with riding hydrogen atoms (excluding the hydrogen atoms on Oγ of serine, O of tyrosine, Oγ1 of threonine, and Nδ1 of histidine), followed by inclusion of the test set data into the refinement. The models were validated with MolProbity (28Davis I.W. Leaver-Fay A. Chen V.B. Block J.N. Kapral G.J. Wang X. Murray L.W. Arendall III, W.B. Snoeyink J. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2007; 35: W375-W383Crossref PubMed Scopus (2981) Google Scholar) and the validation tools in COOT (27Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar). The only Ramachandran plot outlier is Cys106, which is invariably in marginal or outlying regions of the Ramachandran plot in DJ-1 structures.Atomic resolution bond length analysis of residue 18 was performed using unrestrained full matrix least-squares refinement in SHELX-97 as previously described (18Witt A.C. Lakshminarasimhan M. Remington B.C. Hasim S. Pozharski E. Wilson M.A. Biochemistry. 2008; 47: 7430-7440Crossref PubMed Scopus (91) Google Scholar). Estimated standard uncertainties on coordinates and bond lengths were determined by inversion of the blocked (BLOC 1 instruction) full least squares matrix. Final model statistics are provided in Table 1.Mouse Embryonic Fibroblasts (MEFs) and Transfections-Fibroblast cultures were established from wild-type and DJ-1 knock-out mice (29Chandran J.S. Lin X. Zapata A. Höke A. Shimoji M. Moore S.O. Galloway M.P. Laird F.M. Wong P.C. Price D.L. Bailey K.R. Crawley J.N. Shippenberg T. Cai H. Neurobiol. Dis. 2008; 29: 505-514Crossref PubMed Scopus (80) Google Scholar). Expression constructs for human DJ-1 containing a C-terminal V5 tag have been described previously (13Zhong N. Kim C.Y. Rizzu P. Geula C. Porter D.R. Pothos E.N. Squitieri F. Heutink P. Xu J. J. Biol. Chem. 2006; 281: 20940-20948Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Additional mutations (E18N/Q/D and post-V5 stop codon) were generated using QuikChange mutagenesis (Stratagene). Cells were transfected using Lipofectamine 2000 (Invitrogen).Immunocytochemistry and Western Blotting-Mitochondrial fractions were prepared using a commercially available mitochondrial isolation kit (Pierce) as directed. Mitochondrial fractions were then stripped of all loosely associated proteins using 20 μm sodium carbonate in HEPES buffer for 30 min on ice, followed by ultracentrifugation at 60,000 × g for 30 min. Subcellular fractions were Western blotted and probed with a V5-specific antibody (Invitrogen) to visualize transfected DJ-1. Enrichment of mitochondria was confirmed by simultaneously reprobing the same blots with monoclonal antibodies to the voltage-dependent anion channel (VDAC1; Calbiochem clone 31HL, 1:4000) and to cytosolic β-actin (clone AC-15, 1:5000; Sigma). Immunostaining for tagged DJ-1 was performed as described (30Blackinton J. Ahmad R. Miller D.W. van der Brug M.P. Canet-Avilés R.M. Hague S.M. Kaleem M. Cookson M.R. Brain Res. Mol. Brain Res. 2005; 134: 76-83Crossref PubMed Scopus (75) Google Scholar). Paraquat and rotenone were purchased from Sigma.Two-dimensional Gel Electrophoresis-Two-dimensional gel electrophoresis was performed as previously described (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar). Samples were obtained from human M17 neuroblastoma cells that were transiently transfected with various DJ-1 Glu18 mutants. For all DJ-1 constructs, stop codons introduced immediately following the V5 tag were used to avoid changes in pI due to the His6 tag. Immobiline DryStrips (11 cm) with linear separation in the pH range 4-7 (GE Healthcare) were used for isoelectric focusing.Fluorescence Recovery after Photobleaching (FRAP) Measurement of Mitochondrial Fragmentation-FRAP was performed as previously described (31Karbowski M. Norris K.L. Cleland M.M. Jeong S.Y. Youle R.J. Nature. 2006; 443: 658-662Crossref PubMed Scopus (521) Google Scholar, 32Szabadkai G. Simoni A.M. Bianchi K. De Stefani D. Leo S. Wieckowski M.R. Rizzuto R. Biochim. Biophys. Acta. 2006; 1763: 442-449Crossref PubMed Scopus (159) Google Scholar). Cells were transiently transfected with 0.5 mg of mitochondrial matrix-localized yellow fluorescent protein using Fugene and seeded into Lab-Tek borosilicate chambers in phenol red-free Opti-MEM. Circular 25-pixel diameter regions of mitochondria were imaged using an LSM 510 confocal microscope with a Plan-Apochromat ×100/1.4 objective (Zeiss) before and after photobleaching at 100% power with 488- and 514-nm wavelength lasers. Scans were taken in 300-ms intervals, for a total of 40 images over 12 s, and the fluorescence intensity was observed over time. Fluorescence recovery was represented as a fraction of initial fluorescence after normalization to both nonspecific photobleaching and background. Each FRAP curve represented the average of ≥60 measurements over at least two independent experiments. Mobile fractions were calculated as ((FRAPt - background)/FRAPt) × ((NSPBi - background)/NSPBt), where NSPB is the nonspecific photobleaching, the subscript t refers to the signal at time = t and the subscript i refers to the initial signal before photobleaching.Cell Viability-M17 cells were seeded onto glass coverslips and transiently transfected with V5-tagged DJ-1 variants using Lipofectamine 2000 (Invitrogen) for 48 h and then either left untreated or exposed to rotenone (200 nm) for 24 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and stained using monoclonal antibody to V5 (1:500; Invitrogen), followed by anti-mouse IgG conjugated to Alexa-Fluor488 (Molecular Probes). Nuclei were counterstained with Hoechst 33342 (Roche Applied Science), and coverslips were mounted using ProLong gold (Molecular Probes). For each experiment, three randomly selected microscope fields (between 26 and 75 cells/field) were counted by an observer blind to the DJ-1 transfection status of the cells. Each experiment was then repeated three times, and statistical analysis was performed on the combined results. Cell viability was expressed as the percentage of transfected (V5-positive) cells that had intact nuclei compared with all transfected cells. For base line viability in the same cultures, we counted three fields of untransfected cells in the same way, where viability was expressed as percentage of visible nuclei per field.RESULTSSubstitutions at Residue 18 Alter the Oxidation Propensity of Cys106-Three substitutions were made at residue 18 in human DJ-1 for this study; E18Q, E18D, and E18N. The 1.15 Å resolution crystal structure of oxidized E18Q DJ-1 superimposes nearly perfectly (Cα root mean square deviation = 0.08 Å) with wild-type DJ-1 (Protein Data Bank code 1SOA). In addition, like the wild-type protein (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar), E18Q DJ-1 oxidizes during crystal growth to form Cys106-SO2- (Fig. 1B). There are a few small structural differences between E18Q and wild-type DJ-1 near the site of mutation, the most notable being the lengthening of the hydrogen bond between residue 18 and Cys106-SO2- to 2.71 Å (Fig. 1C). In wild-type DJ-1, the 2.47-Å hydrogen bond between the protonated Glu18 carboxylic acid side chain and Cys106-SO2- is an unusually short and presumably a very strong interaction (15Canet-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. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (894) Google Scholar). The 0.24-Å increase in hydrogen bond length in E18Q Cys106-SO2- DJ-1 is accommodated by the correlated displacements of Cys106-SO2- and Gln18 away from each other (Fig. 1C). Because the E18Q substitution is very structurally conservative, altered oxidation for Cys106 in E18Q (see below) can be attributed solely to the changes in the hydrogen bond between residue 18 and Cys106.The crystal structure of oxidized E18N was determined in a previous study and showed that Cys106 is robustly oxidized to" @default.
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• W2063089997 title "Formation of a Stabilized Cysteine Sulfinic Acid Is Critical for the Mitochondrial Function of the Parkinsonism Protein DJ-1" @default.
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• W2063089997 doi "https://doi.org/10.1074/jbc.m806599200" @default.
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