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...

Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry-based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD (very long acyl-CoA dehydrogenase) at Cys238, which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria.

A large number of individuals experience mental health disorders, with cognitive behavioral therapy (CBT) emerging as a standard practice for reduction in psychiatric symptoms, including stress, anger, anxiety, and depression. However, CBT is associated with significant patient dropout and lacks the means to provide objective data regarding a patient’s experience and symptoms between sessions. Emerging wearables and mobile health (mHealth) applications represent an approach that may provide objective data to the patient and provider between CBT sessions. Here, we describe the development of a classifier of real-time physiological stress in a healthy population (n = 35) and apply it in a controlled clinical evaluation for armed forces veterans undergoing CBT for stress and anger management (n = 16). Using cardiovascular and electrodermal inputs from a wearable device, the classifier was able to detect physiological stress in a non-clinical sample with accuracy greater than 90%. In a small clinical sample, patients who used the classifier and an associated mHealth application were less likely to discontinue therapy (p = 0.016, d = 1.34) and significantly improved on measures of stress (p = 0.032, d = 1.61), anxiety (p = 0.050, d = 1.26), and anger (p = 0.046, d = 1.41) compared to controls undergoing CBT alone. Given the large number of individuals that experience mental health disorders and the unmet need for treatment, especially in developing nations, such mHealth approaches have the potential to provide or augment treatment at low cost in the absence of in-person care.