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 metalcatalyzed S-nitrosylation mechanisms. Collectively, the endogenous Snitrosoproteome in the liver has structural features that accommodate multiple mechanisms for selective site-directed S-nitrosylation.cysteine modification | nitric oxide | S-nitrosation | posttranslational modification | proteomics C ysteine S-nitrosylation is a reversible and apparently selective posttranslational protein modification that regulates protein activity, localization, and stability within a variety of organs and cellular systems (1-6). Despite the considerable biological importance of this posttranslational modification, significant gaps exist regarding its in vivo specificity and origin. The identification of in vivo S-nitrosylated proteins has indicated that not all reduced cysteine residues and not all proteins with reduced cysteine residues are modified, implying a biased selection. Several biological chemistries have been proposed to account for the S-nitrosylation of proteins in vivo (1,7,8). Broadly, these include (i) oxidative S-nitrosation by higher oxides of NO, (ii) transnitrosylation by small molecular weight NO carriers such as S-nitrosoglutathione or dinitrosyliron complexes, (iii) catalysis by metalloproteins, and (iv) protein-assisted transnitrosation, as elegantly documented for the S-nitrosylation of caspase-3 by S-nitrosothioredoxin (9, 10). With the exception of the protein-assisted transnitrosylation and metalloprotein catalyzed S-nitrosylation, which we presume necessitates protein-protein interaction, the other proposed mechanisms are rather nondiscriminatory unless th...

S -nitrosation of proteins relevant to Alzheimer’s disease during early stages of neurodegeneration

“…Detection of SNO-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on Cys-150, as previously reported by others using independent approaches, validated the robustness of the protocol (Fig. S1 B and C) (16,29). SNOTRAP also detected previously unidentified SNO-sites, such as SNO-Cys284 of gephyrin (GPHN1), indicating the sensitivity of the method (Fig.…”

“…Despite these observations, many SNO-Cys sites identified in both control and CK-p25 mice did not have charged amino acids within a proximal location of the primary sequence. Previous studies indicate that tertiary structural elements may be required for localizing charged residues to SNOCys sites for many proteins (29,44). Collectively, these data suggest that the specificity for S-nitrosation of a subset of Cys may be dependent on proximal charged amino acids, which possibly provide docking sites for nitrosating and denitrosating agents (29).…”

“…Recent studies indicate that the specificity of SNO-Cys sites may be dependent on the spatial proximity of charged amino acids, which are hypothesized to CK-p25 allow protein-protein interactions that facilitate transnitrsation (29,44). To elucidate common features of SNO-Cys sites, we used the probability LOGO (pLOGO) tool to analyze flanking sequences of SNO-Cys residues for linear motifs (45).…”

On–off system for PI3-kinase–Akt signaling through S -nitrosylation of phosphatase with sequence homology to tensin (PTEN)

“…To determine whether S-nitrosylation affected PTEN activity, we initially monitored recombinant PTEN enzyme activity. Phosphatase activity was evaluated with a standard assay by measuring phosphate released from phosphatidylinositol-3,4,5-trisphosphate [PI (3,4,5) P3], a physiological substrate (23). Exposure of recombinant PTEN to SNOC significantly decreased the level of phosphate in a dose-dependent manner ( Fig.…”

“…This modification occurs by means of oxidative reaction between NO and cysteine (Cys) thiol in the presence of an electron acceptor (such as O 2 or a transition metal) or through transnitrosylation from S-nitrosothiol to another Cys thiol (1)(2)(3). Several methods have been published to detect S-nitrosylated proteins (SNO-Ps) by using antibodies, photolysis, and mercury affinity (4). In particular, the biotin-switch assay is a modified immunoblot developed by Jaffrey and Snyder that has been commonly used to detect endogenous SNO-Ps; this method has greatly advanced the field (5).…”