SummaryProtein S-nitrosylation conveys a large part of the ubiquitous influence of nitric oxide on cellular signal transduction, and accumulating evidence indicates important roles for S-nitrosylation both in normal physiology and in a broad spectrum of human diseases. Here we review recent findings that implicate S-nitrosylation in cardiovascular, pulmonary, musculoskeletal and neurological (dys)function, as well as in cancer. The emerging picture shows that, in many cases, pathophysiology correlates with hypo-or hyper-S-nitrosylation of specific protein targets, rather than a general cellular insult due to loss of or enhanced nitric oxide synthase activity. In addition, it is increasingly evident that dysregulated S-nitrosylation can result not only from alterations in the expression, compartmentalization and/or activity of nitric oxide synthases but can also reflect a contribution from denitrosylases, including prominently the S-nitrosoglutathione (GSNO)-metabolizing enzyme, GSNO reductase. Finally, because exogenous mediators of protein Snitrosylation or denitrosylation can substantially affect the development or progression of disease, potential therapeutic agents that modulate S-nitrosylation could well have broad clinical utility.

No abstract

We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action. Nitric oxide (NO)2 executes a diverse range of cellular functions through the redox-dependent conversion of protein Cys thiols to S-nitrosothiols (SNOs). This post-translational modification, known as S-nitrosylation, has emerged as a highly conserved and spatiotemporally specific signaling mechanism (1). A major contribution to this field was the introduction of the biotin switch technique (BST) by Jaffrey et al. in 2001 (2). Importantly, the BST allows relatively facile identification and quantification of endogenous protein SNOs and thus has greatly contributed to our understanding of protein S-nitrosylation. As evidence, the BST has been employed in over 70 publications, unveiling new roles for S-nitrosylation in events such as apoptosis (3), neurodegeneration (4, 5), insulin signaling (6, 7), and receptor trafficking (8).The BST consists of three principal steps (supplemental Fig. S1): "blocking" of free Cys thiols by S-methylthiolation with S-methyl methanethiosulfonate (a reactive thiosulfonate), formal reduction of SNOs to thiols with ascorbate (Asc), and in situ "labeling" by S-biotinylation of the nascent thiols with biotin-HPDP, a reactive mixed disul...

Protein S-nitrosylation, the post-translational modification of cysteine thiols to form S-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for S-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during physiological and pathophysiological conditions. The focus of the current review is the biotin switch technique (BST), which has become a mainstay assay for detecting S-nitrosylated proteins in complex biological systems. Potential pitfalls and modern adaptations of the BST are discussed, as are future directions for this assay in the burgeoning field of protein S-nitrosylation.

The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALDH ؊/؊ mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NObased cellular signals that may originate independently of NO synthase activity.nitric oxide ͉ nitrite ͉ S-nitrosothiol ͉ nitrate tolerance T he ability of mammalian cells to convert the manmade organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), to vasoactive nitric oxide (NO) or S-nitrosothiol (SNO) played a significant part in the discovery that NO or its equivalent functions as an endogenous physiological mediator (1, 2), and GTN has long served as a principal therapeutic agent for acute angina and congestive heart disease (3-7). Although multiple cellular activities mediating GTN metabolism have been characterized, the mechanisms that specifically subserve GTN bioactivation have remained elusive. Recent evidence indicates a central role for mitochondria in GTN bioactivation. In particular, it has been proposed that the mitochondrial aldehyde dehydrogenase (mtALDH), aldehyde dehydrogenase (ALDH) 2, may provide a principal enzymatic source of GTN-derived NO vasoactivity (through the intermediacy of mitochondrial nitrite) and that mechanism-based mtALDH inactivation contributes to GTN tolerance (8-10). However, this previously uncharacterized role for mitochondria in the generation of NO bioactivity remains unproven, and the centrality of mtALDH in GTN bioactivation in vivo has been disputed (5-7). The findings described in the present study establish that NO bioactivity originating in mitochondria and generated by mtALDH is necessary and sufficient to account for the vasoactivity of clinically relevant concentrations of GTN. Our results suggest, in addition, that inactivation of mtALDH is a principal component of mechanism-based GTN tolerance. Materials and Methods mtALDH ؊/؊ Mice. The generation of mtALDHϪ/Ϫ mice has been described in ref. 11. Wild-type (C57BL͞6) and mtALDH Ϫ/Ϫ mice used for comparative measurements were gender-and agematched (3-4 months). All procedures were approved by the Institutional Animal Care and...

The anaerobic reaction of C. vinosum high-potential iron protein (HiPIP) with nitric oxide has been studied in order to understand the chemical reactivity of NO with protein-bound iron−sulfur clusters. Despite having a solvent inaccessible 4Fe-4S center, native HiPIP reacts with diethylamineNONOate (an NO donor), resulting in protein unfolding and the formation of protein-bound dinitrosyl−iron complexes (DNICs) with a typical g av = 2.03 EPR signal. These cysteinyl-coordinated DNICs are directly observed for the first time by use of electrospray ionization−mass spectrometry (ESI-MS) and are found only in a stoichiometry of 2:1 DNIC:protein. Our results suggest that these complexes form only when the protein is folded. ESI-MS also demonstrates that NO-mediated cluster degradation results in nitrosation of the protein at sites other than cysteine. Finally, reactivity comparison of native and Tyr19Leu HiPIP demonstrates solvent accessibility to be an important, but not necessary factor for Fe−S cluster degradation by NO. By use of UV−visible, NMR, and EPR spectroscopies, as well as ESI-MS, we have determined the major products of degradation and elucidate some of the mechanistic issues governing cluster degradation, protein nitrosation, and DNIC formation. Comparisons are made between the nitric oxide chemistry of bacterial HiPIP and other eukaryotic and prokaryotic iron−sulfur proteins that are relevant in vivo targets for NO.