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Doulias PT , Greene JL , Greco TM , Tenopoulou M , Seeholzer SH , Dunbrack RL , Ischiropoulos H
Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation
Proceedings of the National Academy of Sciences of the United States of America. 2010 Sep;107(39) :16958-16963
PMID: ISI:000282211700042   
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Abstract
S-nitrosylation, the selective posttranslational modification of protein cysteine residues to form S-nitrosocysteine, is one of the molecular mechanisms by which nitric oxide influences diverse biological functions. In this study, unique MS-based proteomic approaches precisely pinpointed the site of S-nitrosylation in 328 peptides in 192 proteins endogenously modified in WT mouse liver. Structural analyses revealed that S-nitrosylated cysteine residues were equally distributed in hydrophobic and hydrophilic areas of proteins with an average predicted pK(a) of 10.01 +/- 2.1. S-nitrosylation sites were over-represented in a-helices and under-represented in coils as compared with unmodified cysteine residues in the same proteins (chi(2) test, P < 0.02). A quantile-quantile probability plot indicated that the distribution of S-nitrosocysteine residues was skewed toward larger surface accessible areas compared with the unmodified cysteine residues in the same proteins. Seventy percent of the S-nitrosylated cysteine residues were surrounded by negatively or positively charged amino acids within a 6-angstrom distance. The location of cysteine residues in a-helices and coils in highly accessible surfaces bordered by charged amino acids implies site directed S-nitrosylation mediated by protein-protein or small molecule interactions. Moreover, 13 modified cysteine residues were coordinated with metals and 15 metalloproteins were endogenously modified supporting metal-catalyzed S-nitrosylation mechanisms. Collectively, the endogenous S-nitrosoproteome in the liver has structural features that accommodate multiple mechanisms for selective site-directed S-nitrosylation.
Notes
Doulias, Paschalis-Thomas Greene, Jennifer L. Greco, Todd M. Tenopoulou, Margarita Seeholzer, Steve H. Dunbrack, Roland L. Ischiropoulos, Harry National Institutes of Health [AG13966, HL054926]; National Institute of Environmental Health Sciences Center of Excellence in Environmental Toxicology [ES013508]; National Institute of General Medical Sciences [F31GM085903] We thank the Protein Core at the Children's Hospital of Philadelphia Research Institute for their assistance with mass spectrometry, Dr. Santosh S. Venkatesh for assistance with the. 2 test, Dr. David Schwartz (Solulink Biosciences, San Diego, CA) for the synthesis of organomercury- polyethyleneglycol-biotin, and Dr. Qi Fang for support with structural analysis. This work was supported by National Institutes of Health Grants AG13966 and HL054926, National Institute of Environmental Health Sciences Center of Excellence in Environmental Toxicology Grant ES013508 (to H.I), and National Institute of General Medical Sciences Award F31GM085903 (to J.L.G.). H.I. is the Gisela and Dennis Alter Research Professor of Pediatrics. 42 Natl acad sciences; 2101 constitution ave nw, washington, dc 20418 usa 655aa