Protein S-Glutathiolation Triggered by DecomposedS-Nitrosoglutathione

Abstract

Recombinant human brain calbindin D(28K) (rHCaBP), human Cu,Zn-superoxide dismutase (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed S-nitrosoglutathione (GSNO) solutions. Tryptic or Glu-C digestion and MALDI-TOF MS analyses of the digests are consistent with S-thiolation of Cys111 and Cys187 of HCuZnSOD and rHCaBP, respectively, upon exposure to decomposed GSNO. GAPDH activity analysis reveals that S-glutathiolation most likely occurs on the active site Cys149, and the single free Cys34 is assumed to be the site of S-glutathiolation in BSA. The yields of S-glutathiolation of rHCaBP, GAPDH, and BSA were much higher than those of HCuZnSOD. The latter is limited by the accessibility of Cys111 to the glutathiolating reagent in the HCuZnSOD dimer. Unlike decomposed GSNO, fresh GSNO, reduced glutathione (GSH), and oxidized glutathione (GSSG) are not efficient S-glutathiolating agents for the proteins examined here. On the basis of analysis by mass spectrometry and UV-visible absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO(2)SG), and GSSG as products. GS(O)SG is the efficient protein S-glutathiolating agent in GSNO solutions, not GSNO, which does not carry out efficient S-glutathiolation of rHCaBP, HCuZnSOD, or GAPDH in vitro. A hydrolysis pathway yielding GSOH and nitroxyl (HNO/NO(-)) as intermediates is proposed for GSNO decomposition in the dark. This is based on inhibition of GSNO breakdown by dimedone, a reagent specific for sulfenic acids, and on nitroxyl scavenging by metmyoglobin. The results presented here are contrary to numerous reports of protein S-thiolation by low-molecular weight S-nitrosothiols.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

“…A further transnitrosation step with GSH can regenerate GSNO (61). Previous studies have shown that GSNO is not a good glutathionylating agent in vitro (54,55), and our data (not shown) indicated that it is not a glutathionylating substrate for GSTO1-1 in the peptide fluorescence assay. Thus the effect of GSTO1-1 on the protein glutathionylation that occurs when cells are treated with GSNO appears to be indirect.…”

“…These results suggest that under these conditions GSTO1-1 acts primarily as a deglutathionylating enzyme. (54,55), it has previously been shown to strongly induce glutathionylation in cultured cells (44,56). In this study we examined the time course of intracellular glutathionylation after treatment with 1 mM GSNO and compared T47-D cells transfected with either an empty pCDNA3 plasmid vector, cells transfected with pCDNA3-GSTO1C32A, or cells transfected with pCDNA3-GSTO1.…”

S-nitrosoglutathione inhibits α1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery

“…The reactive nitrogen species peroxynitrite also promotes S-glutathionylation of the sarco/endoplasmic reticulum calcium ATPase in the presence of GSH to mediate aortic relaxation (1). Recent data also indicate that glutathione sulfoxide, a GSNO decomposition product, rather than GSNO, can promote mixed disulfide formation (29). We did not exclude the possibility that these other cysteine modifications may also occur in the pulmonary circulation (11,16).…”

The kinetics of thiol-mediated decomposition of S-nitrosothiols

“…[1][2][3][4][5][6][7][8][9] Numerous studies have suggested that RSNO is the carrier, or the storage form, of NO in vivo, thereby serving as a reservoir for NO bioactivity. [10][11][12][13][14][15] The study of chemical stability of RSNO therefore provides basic information about RSNO as potential NO carriers or donors.…”