Nitrosothiol Reactivity Profiling Identifies S-Nitrosylated Proteins with Unexpected Stability

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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...

Section: Resultsmentioning

confidence: 94%

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation

Doulias

Greene

Greco

et al. 2010

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306

“…Accordingly, in most instances, the time course of these processes might not be rate limiting for the overall events that comprise NMDAR activation, stimulation of nNOS to generate NO, nitrosylation of stargazin, and its translocation along with GluR1. However, in some cases, both basal and stimulated denitrosylation take place over varied time scales (20)(21)(22)(23)(24). In the case of stargazin-mediated GluR1 surface expression, nitrosylation of stargazin does not have an obligate role in trafficking; instead, it exerts a regulatory influence.…”

Section: Discussionmentioning

confidence: 99%

S-nitrosylation of stargazin regulates surface expression of AMPA-glutamate neurotransmitter receptors

Selvakumar

Huganir

Snyder

2009

Synaptic plasticity is mediated by changes in the surface expression of AMPA receptors (AMPARs). Stargazin and related transmembrane AMPAR regulatory proteins have emerged as the principal regulators of AMPAR surface expression. Here, we show in heterologous cells and primary neurons that stargazin is physiologically S-nitrosylated, resulting in increased surface expression. Snitrosylation of stargazin increases binding to the AMPAR subunit GluR1, causing increased surface expression of the AMPAR. NMDAR stimulation, well known to activate neuronal nitric oxide synthase, increases both nitrosylation of stargazin and its binding to AMPAR. Thus, S-nitrosylation of stargazin is a physiologic regulator of AMPAR surface expression.

“…S-nitrosylated proteins were assayed by the biotin substitution as previously described (28). CFTR IP (noted previously) was carried out on each fraction at each time, followed by biotin substitution, streptavidin isolation, and IB for Hop.…”

Section: Methodsmentioning

confidence: 99%

“…Role of Hop Cysteine 403 in Hop Degradation. Hop has a cysteine (C403) in a motif that can be associated with S-nitrosylation, particularly under conditions of inflammation (26,28). Overexpression of C→S mutation of Hop Cys-403 resulted in (i) protection from GSNO-induced Hop degradation and (ii) decreased Hop S-nitrosylation C403S relative to expression by GSNO (Fig.…”

Section: Gsno Decreases Hop Expression In Different Cellmentioning

confidence: 99%

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

Marozkina¹,

Yemen²,

Borowitz³

et al. 2010

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product ΔF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in ΔF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of ΔF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on ΔF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects ΔF508 CFTR trafficking by inhibiting Hop expression, and that combination therapiesusing differing mechanisms of action-may have additive benefits in treating CF.cystic fibrosis transmembrane conductance regulator | S-nitrosoglutathione corrector | treatment