Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells

S-nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. Even though nitric oxide is intimately involved in the regulation of vascular smooth muscle cell functions, the potential protein targets for nitric oxide modification as well as structural features that underlie the specificity of protein S-nitrosocysteine formation in these cells remain unknown. Therefore, we used a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins including cytoskeletal proteins, chaperones, proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid/base motifs, as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle.     

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

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 (χ² 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.     

Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.     

S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity

Endothelial nitric oxide synthase (eNOS) is active only as a homodimer. Recent data has demonstrated that exogenous NO can act as an inhibitor of eNOS activity both in intact animals and vascular endothelial cells. However, the exact mechanism by which NO exerts its inhibitory action is unclear. Our initial experiments in bovine aortic endothelial cells indicated that exogenous NO decreased NOS activity with an associated decrease in eNOS dimer levels. We then undertook a series of studies to investigate the mechanism of dimer disruption. Exposure of purified human eNOS protein to NO donors or calcium-mediated activation of the enzyme resulted in a shift in eNOS from a predominantly dimeric to a predominantly monomeric enzyme. Further studies indicated that endogenous NOS activity or NO exposure caused S-nitrosylation of eNOS and that the presence of the thioredoxin and thioredoxin reductase system could significantly protect eNOS dimer levels and prevent the resultant monomerization and loss of activity. Further, exogenous NO treatment caused zinc tetrathiolate cluster destruction at the dimer interface. To further determine whether S-nitrosylation within this region could explain the effect of NO on eNOS, we purified a C99A eNOS mutant enzyme lacking the tetrathiolate cluster and analyzed its oligomeric state. This enzyme was predominantly monomeric, implicating a role for the tetrathiolate cluster in dimer maintenance and stability. Therefore, this study links the inhibitory action of NO with the destruction of zinc tetrathiolate cluster at the dimeric interface through S-nitrosylation of the cysteine residues.     

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

Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.     

Nitric oxide signaling in the cardiovascular system: implications for heart failure

PURPOSE OF REVIEW: The role played by nitric oxide (NO) in cardiovascular physiology remains highly controversial. Following the discovery that NO is the prototypic endothelium-derived relaxing factor, this signaling molecule was implicated as possessing many other biological actions within the cardiovascular system, including effects on cardiac contraction, relaxation, and energetics. Here, we discuss new concepts regarding NO signaling, its effector pathways, and interactions between NO and the redox milieu within a framework of cardiac physiology and pathophysiology. RECENT FINDINGS: Major recent insights that have advanced understanding of the mechanisms of NO bioactivity include the following. (1) NO acts in subcellular signaling compartments or modules. (2) S-nitrosylation (covalent modification of cysteine thiol moieties) of proteins represents a prototypic second messenger signaling mode in biologic systems. (3) Reactive oxygen and nitrogen species work together to facilitate signaling. (4) Disruption of physiologic signaling can occur by either increased formation of reactive oxygen species or decreased production of reactive nitrogen species, a situation of nitroso-redox imbalance. SUMMARY: These insights, which challenge classically held views that NO acts as a freely diffusible molecule regulated primarily by concentration and exerting signaling primarily through cyclic GMP production, offer a new perspective on the pathophysiology and treatment of congestive heart failure.     

Inducible NO synthase dependent S-nitrosylation and activation of arginase1 contribute to age-related endothelial dysfunction

Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, L-arginine. However, the high Km of arginase (>1 mmol/L) compared with NOS (2 to 20 micromol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for L-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its Km value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its Km decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for L-arginine and contribute to endothelial dysfunction in the aging cardiovascular system.     

Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation

Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 micromol/l of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 micromol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First, we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl2. Second, S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third, dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes.     

Balancing reactivity against selectivity: the evolution of protein S-nitrosylation as an effector of cell signaling by nitric oxide

Produced by the action of lightning in the atmosphere of the pre-biotic earth, nitric oxide (NO) is a free radical molecule that provided the major nitrogen source for development of life. Remarkably, when atmospheric sources of NO became restrictive, organisms evolved the capacity for NO biosynthesis and NO took on bioregulatory roles. We now recognize NO as an ancestral regulator of diverse and important biological functions, acting throughout the phylogenetic tree. In mammals, NO has been implicated as a pivotal regulator of virtually every major physiological system. The bioactivities of NO, and reactive species derived from NO, arise predominantly from their covalent addition to proteins. Importantly, S-nitrosylation of protein cysteine (Cys) residues has emerged as a preeminent effector of NO bioactivity. How and why NO selectively adds to particular Cys residues in proteins is poorly understood, yet fundamental to how NO communicates its bioactivities. Also, evolutionary pressures that have shaped S-nitrosylation as a biosignaling modality are obscure. Considering recently recognized NO signaling paradigms, we speculate on the origin of NO signaling in biological systems and the molecular adaptations that have endowed NO with the ability to selectively target a subset of protein Cys residues that mediate biosignaling.     

S-Nitrosylation activates Cdk5 and contributes to synaptic spine loss induced by beta-amyloid peptide

The activity of Cdk5 and its regulatory subunit p35 is thought to be important in both normal brain function and neurodegenerative disease pathogenesis. Increased Cdk5 activity, via proteolytic cleavage of p35 to a p25 fragment by the calcium-activated protease calpain or by phosphorylation at Cdk5(Tyr15), can contribute to neurotoxicity. Nonetheless, our knowledge of regulation of Cdk5 activity in disease states is still emerging. Here we demonstrate that Cdk5 is activated by S-nitrosylation or reaction of nitric oxide (NO)-related species with the thiol groups of cysteine residues 83 and 157, to form SNO-Cdk5. We then show that S-nitrosylation of Cdk5 contributes to amyloid-β (Aβ) peptide-induced dendritic spine loss. Furthermore, we observed significant levels of SNO-Cdk5 in postmortem Alzheimer's disease (AD) but not in normal human brains. These findings suggest that S-nitrosylation of Cdk5 is an aberrant regulatory mechanism of enzyme activity that may contribute to the pathogenesis of AD.     

Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation

The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration- and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the alpha- and beta-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in alpha and C122 in beta, created mutants that were mostly resistant to desensitization. Structural analysis of the region near beta-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.     

Signalling by NO-induced protein S-nitrosylation and S-glutathionylation: convergences and divergences

The role of nitric oxide in several signalling routes has been clearly established. In recent years increasing attention has been paid to its ability to produce covalent protein post-translational modifications in conjunction with other reactive oxygen and nitrogen species. Among these, the modification of cysteine residues has been shown to be of particular importance due to the functional relevance of many of them. In this review, we focus on the modification of the cysteine thiol by incorporation of a NO moiety (S-nitrosylation) or of a glutathione moiety (S-glutathionylation). Both modifications are produced by different reactions induced by nitric oxide-related species. We discuss the differences and similarities of both modifications, and their relationships, in regard to the biochemical mechanisms that produce them, including the enzymatic activities that may catalyze some of them and their subcellular compartmentalization. Even when biochemical knowledge is one step ahead of the demonstration of their pathophysiological relevance, we also describe the potential role of both modifications in several processes in which both post-translational modifications are involved.     

SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures

Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) Nat. Cell. Biol. 3, 193-197], comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides, and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S-nitrosoglutathione-treated (2-10 microM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including alpha-tubulin, beta-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S-nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation.     

S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu catalytic triad [Murray-Rust, J., Leiper, J. M., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P. & McDonald, N. (2001) Nat. Struct. Biol. 8, 679-683]. The presence of a reactive cysteine residue (Cys-249) in the active site of DDAH raised the possibility that DDAH activity might be directly regulated by S-nitrosylation of this residue by NO. In the present study, we demonstrate that recombinant DDAH is reversibly inhibited after incubation with NO donors in vitro. Similarly mammalian DDAH in cytosolic extracts is also reversibly inhibited by NO donors. In cultured endothelial cells, heterologously expressed human DDAH II was S-nitrosylated after cytokine induced expression of the inducible NOS isoforms. The implication of these findings is that under certain conditions when NO generation increases, S-nitrosylation diminishes DDAH activity and this would be expected to lead to accumulation of asymmetric dimethylarginine and inhibition of NOS. This observation may help explain why expression of iNOS often leads to inhibition of activity of constitutively expressed NOS isozymes. We also identify Cys-His-Glu as a nitrosylation motif that is conserved in a family of arginine handling enzymes.     

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.     

Nitric oxide regulates endocytosis by S-nitrosylation of dynamin

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the beta(2) adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both beta(2) adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both beta(2) adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.