Bauer, Joseph Alan, Jourd'heuil, Frances L., Lowery, Anthony M., Melton, Elaina M., Mnaimneh, Sanie, Bryan, Nathan S., Fernandez, Bernadette O., Park, Joo-Ho, Ha, Chung-Eun, Bhagavan, Nadhipuram V., Feelisch, Martin and Jourd'heuil, David. (2010) Redox-sensitivity and site-specificity of S- and N- denitrosation in proteins. PLoS One, Vol.5 (No.12). e14400. ISSN 1932-6203

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Abstract

Background S-nitrosation – the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins – is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. Methodology/Principal Findings The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. Conclusions We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.

Item Type:	Journal Article
Subjects:	R Medicine > R Medicine (General)
Divisions:	Faculty of Medicine > Warwick Medical School > Metabolic and Vascular Health Faculty of Medicine > Warwick Medical School
Journal or Publication Title:	PLoS One
Publisher:	Public Library of Science
ISSN:	1932-6203
Date:	21 December 2010
Volume:	Vol.5
Number:	No.12
Page Range:	e14400
Identification Number:	10.1371/journal.pone.0014400
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
URI:	http://wrap.warwick.ac.uk/id/eprint/46376

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