S-Nitrosylation and S-Glutathiolation of Protein Sulfhydryls byS-Nitroso Glutathione

Ji, Yin; Akerboom, Theodorus P.M.; Sies, Helmut; Thomas, James A.

doi:10.1006/abbi.1998.1013

Cited by 173 publications

(118 citation statements)

References 38 publications

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“…It is likely that S-thiolation occurs at the reactive and catalytically essential cysteine 149 (34). Oxidation of this residue inactivates the enzyme and may be achieved by a range of oxidants including GSSG, GSNO, NO, HOCl, and GS(O)SG (33,(35)(36)(37)(38)(39)(40). Triosephosphate isomerase is another glycolytic enzyme that we found to be S-thiolated during cardiac oxidative stress.…”

Section: S-thiolation During Cardiac Oxidant Stress and Functionalmentioning

confidence: 90%

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Eaton¹,

Byers²,

Leeds³

et al. 2002

Journal of Biological Chemistry

167

133

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We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3-and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase C␣, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.

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Section: S-thiolation During Cardiac Oxidant Stress and Functionalmentioning

confidence: 90%

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Eaton¹,

Byers²,

Leeds³

et al. 2002

Journal of Biological Chemistry

167

133

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show abstract

“…Numerous classes of proteins contain free cysteine residues that are highly conserved across species, suggesting regulatory possibilities, beyond structural roles and metal ion coordination. In addition, cysteine residues can be modified through alternative redoxbased modifications (SNO, SOH, SSG, S-S) that may enable differential effects on protein function [32][33][34][35]. In other words, distinct reversible modifications of cysteines ( Figure 1) may lead to unique functional outcomes ( Figure 2) as well as provide a mechanism through which differential responsitivity can be achieved.…”

Section: Oxidizable Amino Acids: Reactive Cysteines As Redox Targetsmentioning

confidence: 99%

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

Janssen–Heininger¹,

Mossman²,

Heintz³

et al. 2008

Free Radical Biology and Medicine

699

652

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“…Discussion NO donors like GSNO or SNAP have the potential to induce S-nitrosylation in proteins ( Ji et al, 1999;Mallis and Thomas, 2000). Detection of S-nitrosylated proteins is commonly done by chemical assays like triiodide chemiluminescence and photolytic chemiluminescence (Stamler et al, 1992;Eu et al, 2000).…”

Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 99%

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that Snitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975=990, and 1047=1049 as being susceptible to Snitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

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S-Nitrosylation and S-Glutathiolation of Protein Sulfhydryls byS-Nitroso Glutathione

Cited by 173 publications

References 38 publications

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

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