title: Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation.
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 α-helices and under-represented in coils as compared with unmodified cysteine residues in the same proteins (χ(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-Å distance. The location of cysteine residues in α-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.
authors.affiliation: Children's Hospital of Philadelphia Research Institute and Department of Pharmacology, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Paschalis-Thomas
authors.initials: PT
authors.lastname: Doulias
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Greene
authors.forename: Todd M
authors.initials: TM
authors.lastname: Greco
authors.forename: Margarita
authors.initials: M
authors.lastname: Tenopoulou
authors.forename: Steve H
authors.initials: SH
authors.lastname: Seeholzer
authors.forename: Roland L
authors.initials: RL
authors.lastname: Dunbrack
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics; D026403:S-Nitrosothiols / Q000032:analysis / Q000378:metabolism*
chemical_list: D011506:Proteins; D020543:Proteome; D026403:S-Nitrosothiols; C021315:S-nitrosocysteine; D003545:Cysteine