title: Proteomic profiling of nitrosative stress: protein S-oxidation accompanies S-nitrosylation.
abstract: Reversible chemical modifications of protein cysteine residues by S-nitrosylation and S-oxidation are increasingly recognized as important regulatory mechanisms for many protein classes associated with cellular signaling and stress response. Both modifications may theoretically occur under cellular nitrosative or nitroxidative stress. Therefore, a proteomic isotope-coded approach to parallel, quantitative analysis of cysteome S-nitrosylation and S-oxidation was developed. Modifications of cysteine residues of (i) human glutathione-S-transferase P1-1 (GSTP1) and (ii) the schistosomiasis drug target thioredoxin glutathione reductase (TGR) were studied. Both S-nitrosylation (SNO) and S-oxidation to disulfide (SS) were observed for reactive cysteines, dependent on concentration of added S-nitrosocysteine (CysNO) and independent of oxygen. SNO and SS modifications of GSTP1 were quantified and compared for therapeutically relevant NO and HNO donors from different chemical classes, revealing oxidative modification for all donors. Observations on GSTP1 were extended to cell cultures, analyzed after lysis and in-gel digestion. Treatment of living neuronal cells with CysNO, to induce nitrosative stress, caused levels of S-nitrosylation and S-oxidation of GSTP1 comparable to those of cell-free studies. Cysteine modifications of PARK7/DJ-1, peroxiredoxin-2, and other proteins were identified, quantified, and compared to overall levels of protein S-nitrosylation. The new methodology has allowed identification and quantitation of specific cysteome modifications, demonstrating that nitroxidation to protein disulfides occurs concurrently with S-nitrosylation to protein-SNO in recombinant proteins and living cells under nitrosative stress.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, University of Illinois at Chicago , 833 S. Wood Street, Chicago, Illinois 60612-7231, United States.
authors.forename: Yue-Ting
authors.initials: YT
authors.lastname: Wang
authors.forename: Sujeewa C
authors.initials: SC
authors.lastname: Piyankarage
authors.forename: David L
authors.initials: DL
authors.lastname: Williams
authors.forename: Gregory R J
authors.initials: GR
authors.lastname: Thatcher
mesh_terms: D045744:Cell Line, Tumor; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D051549:Glutathione S-Transferase pi / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry; D020030:Nitric Oxide Donors / Q000737:chemistry; D015538:Nitrosation; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D026403:S-Nitrosothiols / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D004220:Disulfides; D009097:Multienzyme Complexes; D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; D009569:Nitric Oxide; C021315:S-nitrosocysteine; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D003545:Cysteine