Trends in Molecular Medicine
ReviewFeature ReviewProtein S-nitrosylation in health and disease: a current perspective
Introduction
In mammalian cells, the L-Arg-dependent nitric oxide (NO) synthases – neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2) and endothelial NOS (eNOS, NOS3) – are the major sources of endogenous NO, and stimulus-coupled activation or induction of NO synthases has been shown to mediate or modulate a broad range of cellular signaling pathways. The physiological influence of NO is exerted predominantly through the posttranslational modification and functional regulation of proteins. It was first established that nitrosylation of heme iron within soluble guanylate cyclase activates the enzyme to generate cyclic GMP and thereby subserves NO-based vasoactivity. However, hemes do not generally elicit cellular signaling involving posttranslational modification of proteins and thus an explanation for most NO-based bioactivity was not apparent. Subsequently, a large body of experimental evidence has demonstrated that S-nitrosylation of Cys residues within a broad functional spectrum of proteins constitute a large part of the ubiquitous influence of NO on cellular function [1].
Expression of iNOS is induced in many mammalian cell types by a variety of stressors or injury and the cytotoxic action of NO, generated in particular by phagocytic cells, raised the possibility that NO generated at relatively high and sustained levels by iNOS could compromise cellular function through generalized nitrosative stress. However, the emerging recognition that NO is involved in a multiplicity of cellular signal transduction pathways through protein S-nitrosylation pointed to the possibility that dysregulated S-nitrosylation could contribute to pathophysiologies characteristic of a wide range of disease states [2]. The relatively recent development of improved methods for analysis of protein S-nitrosylation 3, 4 has facilitated the identification of numerous S-nitrosylated proteins (SNO-proteins) for which levels of S-nitrosylation can be altered in disease. The emerging picture shows that hypo- or hyper-S-nitrosylation of these specific protein targets (which result in alterations in protein function) are directly implicated in the etiology and symptomatology of an increasing number of human diseases, prominently including disorders of the cardiovascular, musculoskeletal and nervous systems (Table 1). In a number of cases, specific Cys residues that are the loci of (patho)physiological regulation by S-nitrosylation have been identified.
The molecular mechanisms underlying (dys)regulation of S-nitrosylation and possible approaches to therapeutic alteration of SNO-protein levels are now the focus of increasing attention. Although NOS expression and activity are obvious governors of S-nitrosylation, the co-localization of NOS enzymes with target proteins, including direct interactions, seems in many cases to be an important determinant of S-nitrosylation under physiological conditions; accordingly, aberrant NOS localization seems to be involved in at least several diseases (Figure 1). Such deficits can have a genetic basis. For example, in a variant of long QT syndrome, a mutation in an nNOS scaffold protein results in disinhibition of nNOS and aberrant S-nitrosylation of a cardiac ion channel [5]. In addition to localization, it has been established that transfer of NO groups between proteins and glutathione governs a cellular equilibrium between low-molecular-weight and protein S-nitrosothiols (SNOs) (Figure 2). In mouse models, genetic ablation of S-nitrosoglutathione reductase (GSNOR), the enzyme principally responsible for GSNO metabolism, results in enhanced levels of SNO-proteins and significantly attenuates experimental asthma and heart failure 6, 7, but increases the severity of endotoxic shock [8]. Finally, the therapeutic potential of agents that affect S-nitrosylation is being explored with promising results (Table 2). These agents have the potential to restore deficient SNO-proteins to physiological levels or to otherwise influence cellular signaling pathways that are mediated or modulated by S-nitrosylation. For example, SNO-repleting agents are highly efficacious in the setting of inflammation, including experimental models of lung injury, stroke and multiple sclerosis, in which S-nitrosylation seems to play a major role in expression of the innate immune response 9, 10, 11, 12. This review focuses on the numerous proteins and signaling pathways that are regulated by S-nitrosylation in the context of diseases, in which aberrant S-nitrosylation has recently been implicated.
Section snippets
Modulation of SNO production: NO synthases and organic nitrates
In tissues and extracellular fluids, SNO levels are likely to reflect NOS activity and, accordingly, can be modulated through altered expression or activity of enzymes that control the availability of endogenous NOS substrates (e.g. L-Arg) or endogenous NOS inhibitors (e.g. asymmetric dimethylarginine, ADMA). In addition, a number of enzymes in the L-Arg/NO pathway are also targets of regulatory S-nitrosylation [1], including arginase, which catabolizes L-Arg. Arginase 1 (Arg1; cytosolic
GSNO reductase and its regulation by NO/SNO: implications for asthma
A major mechanism for protein S-nitrosylation in vivo is the reversible transnitrosylation of protein thiols by GSNO, the predominant low-molecular-weight SNO (Figure 2) [2]. GSNO is the preferred physiological substrate for glutathione-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase, ADH III, which in methylotropic bacteria can also metabolize formaldehyde), whereas alcohols are apparently not physiological substrates of ADH III. Thus, the enzyme has been renamed GSNO
Ras S-nitrosylation: adaptive immunity and tumor maintenance
Many GTPases within the Ras superfamily contain redox-sensitive Cys residues that are susceptible to S-nitrosylation [34]. In the cases of H-, K- and N-Ras, NO promotes the conversion of inactive GDP-bound Ras to its active GTP-bound form by guanine nucleotide exchange. Ras activation is coincident with S-nitrosylation of C118, which resides within the nucleotide-binding domain; mutation of C118 abolishes NO-induced Ras activation [34]. Although S-nitrosylation of Ras is closely coupled to NOS
Aberrant hemoglobin S-nitrosylation and the human respiratory cycle: sickle cell anemia, banked blood and pulmonary arterial hypertension
Accumulating evidence supports a role for red blood cell (RBC) SNOs, which originate from S-nitroso-(βCys93)hemoglobin (SNO-Hb), in mediating oxygen tension (PO2)-dependent vasodilatory activity [43] in the human respiratory cycle. In the three-gas (NO, O2, CO2) model of the respiratory cycle, RBCs liberate NO-based bioactivity to enable efficient O2 delivery (which is primarily a function of blood flow). This activity of RBCs seems to require the transfer of SNO from Hb to RBC
Control of hypoxic signaling: tumor radiotherapy, protection from myocardial ischemia, and development of pulmonary arterial hypertension
HIF-1, a master transcriptional regulator, is activated at low oxygen tension through stabilization of its α subunit (HIF-1α). Under normoxia, hydroxylation of HIF-1α by an O2-dependent prolyl hydroxylase promotes its binding with and ubiquitylation by the E3 ligase complex containing Von Hippel-Lindau disease tumor suppressor (pVHL) and subsequent proteasomal degradation. Accumulating evidence suggests that, under normoxia, NO/SNO stabilizes HIF-1α (thus mimicking hypoxia) [1].
S
(Dys)regulated S-nitrosylation in the heart through differential NOS expression and localization: contractility, ischemia and long QT
The importance of NOS subcellular compartmentalization is well exemplified in the heart, where eNOS and nNOS are differentially localized and exhibit opposite effects on myocardial contractility 25, 59, 60. eNOS, associated primarily with sarcolemmal caveolae, attenuates β-AR-dependent myocardial contractility [61]; inhibition of the L-type Ca2+ channel (LTCC), at least in part by S-nitrosylation [26], seems to underlie this effect. By contrast, nNOS is primarily localized to the sarcoplasmic
S-nitrosylation in disorders of skeletal muscle
The skeletal muscle ryanodine receptor (RyR) isoform RyR1 is activated by S-nitrosylation of C3635, which reduces the inhibitory effect of Ca2+–CaM on the channel [74]. A number of mutations in RyR1 (e.g. Y522S) are associated with malignant hyperthermia and related diseases that are characterized by involuntary muscle contractures, tissue lysis (rhabdomyolysis) and sudden death in response to elevated environmental temperatures 75, 76. Heterozygous RyR1Y522S/wt mutant mice, which have a
iNOS-induced S-nitrosylation in diabetes
Accumulating evidence links insulin resistance in type 2 diabetes to NO production and protein S-nitrosylation [83]. iNOS expression is increased in mouse models of diabetes and iNOS knockout mice fed a high-fat diet develop obesity but have improved glucose tolerance and normal insulin sensitivity (in vivo) and insulin-stimulated glucose uptake (ex vivo) compared with diet-induced obese wild-type mice [84]. iNOS-dependent S-nitrosylation of protein kinase B (PKB/Akt), the insulin receptor
Nitrosative stress in neurodegenerative diseases
Dysregulated protein S-nitrosylation (which can result from overactivation of NMDA receptors) seems to be prevalent in neurodegenerative disorders characterized, in particular, by the accumulation of misfolded proteins 2, 93. Hyper-S-nitrosylation of parkin, protein disulfide isomerase (PDI), peroxiredoxin 2 (Pdx2), X-linked inhibitor of apoptosis (XIAP) and dynamin-related protein 1 (Drp1) is observed in brains from patients with neurological disorders (including Alzheimer's, sporadic
SNO-GAPDH and cell death: therapeutic implications in neurodegeneration and cancer
Nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a hallmark of stress-induced cell death [100]. Stimulation of nNOS in neurons or of iNOS in macrophages (by NMDA and cytokines, respectively) triggers GAPDH S-nitrosylation at its active site, C145, which promotes GAPDH association with the E3 ubiquitin ligase Siah1 and results in nuclear translocation of the SNO-GAPDH–Siah1 complex [101]. Within the nucleus, SNO-GAPDH stabilizes Siah1, which facilitates the
Regulation of prostaglandin synthesis: preconditioning in ischemia–reperfusion injury
Prostaglandin synthesis by cyclo-oxygenase (COX) is coupled to iNOS activation in cells and tissues and S-nitrosylation is implicated in this effect. iNOS binds directly to and activates COX-2 by S-nitrosylation of a single cysteine (C526) proximal to the substrate (arachidonic acid, AA) binding site [112] and COX-2 S-nitrosylation and prostaglandin E2 formation are attenuated by blocking iNOS–COX-2 interaction [112]. Similarly, binding and S-nitrosylation of COX-2 by nNOS seem to mediate NMDA
Anti-inflammatory activities of endogenous and exogenous S-nitrosothiols
Protein S-nitrosylation by iNOS-derived NO occurs downstream of TLR activation and can be proinflammatory; targets of TLR-dependent S-nitrosylation include surfactant protein D, which is increasingly implicated in lung inflammation 122, 123. However, numerous additional studies point to a role for S-nitrosylation in the feedback inhibition of TLR-mediated signaling. NOS and/or low-molecular-weight SNOs inhibit the expression both of iNOS 124, 125, 126 and of multiple cytokines {interleukin-1β
Future directions
The broad purview of protein S-nitrosylation in normal and disturbed cell function presents, in principle, novel therapeutic opportunities in a wide range of human diseases. These opportunities remain largely untapped. In addition, the improvement and dissemination of new methodologies for analysis of protein S-nitrosylation – which have facilitated many of the recent discoveries detailed here and have been reviewed recently 137, 138 – will allow the role of aberrant S-nitrosylation to be
Disclosure statement
JSS holds equity in LifeHealth, N30 Pharma and Vindica, companies developing assays and uses for NO-based molecules.
Acknowledgements
This work was supported by grants U19-ES012496, HL075443 and HL059130 from the NIH.
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