S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.

It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-kappaB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation.

A growing body of evidence suggests that the cellular response to oxidative and nitrosative stress is primarily regulated at the level of transcription. Posttranslational modification of transcription factors may provide a mechanism by which cells sense these redox changes. In bacteria, for example, OxyR senses redox-related changes via oxidation or nitrosylation of a free thiol in the DNA binding region. This mode of regulation may serve as a paradigm for redox-sensing by eukaryotic transcription factors as most-including NF-kappaB, AP-1, and p53-contain reactive thiols in their DNA binding regions, the modification of which alters binding in vitro. Several of these transcription factors have been found to be sensitive to both reactive oxygen species and nitric oxide-related species in vivo. It remains entirely unclear, however, if oxidation or nitrosylation of eukaryotic transcription factors is an important mode of regulation, or whether transcriptional activating pathways are principally controlled at other redox-sensitive levels.-Marshall, H. E., Merchant, K., Stamler, J. S. Nitrosation and oxidation in the regulation of gene expression.

Signal transduction in the NF-B transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-B p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-B p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-B-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-B binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-B activity and regulates gene expression in inflammation.The transcription factor NF-B controls the expression of many genes involved in the inflammatory response (1). One of these genes is the inducible nitric oxide synthase (NOS2) 2 whose activity impacts the cellular response to acute injury (2). The product of NOS2, nitric oxide (NO), is known to modulate NF-B activity at multiple steps in the signal transduction pathway (3). The primary molecular mechanism by which NO alters NF-B signaling is via S-nitrosylation, with several different NF-B proteins including IB kinase ␤ and p50 regulated by this post-translational modification (4, 5). Particularly, we have shown that p50 is S-nitrosylated under conditions of nitrosative stress and is associated with a decrease in NF-B (p50-p65) DNA binding (4). However, the physiological significance of S-nitrosylation of the NF-B p50-p65 heterodimer in the context of cytokine signaling and cellular NOS2 expression has not been established.NOS2 expression is dependent upon NF-B activation, with the cytokine-responsive B-binding site(s) identified in both the human and the murine NOS2 promoters (6, 7). Cytokinestimulated NOS2 activity, in turn, inhibits NF-B-dependent transcription, but the specific molecular target(s) of NOS2 in the NF-B pathway have not been elucidated (8). We have previously demonstrated that cytokine-induced NOS activity inhibits NF-B DNA binding in a reversible manner, a mechanism consistent with S-nitrosylation of the p50-p65 heterodimer (4). Moreover, evidence accumulated recently suggests a central role of S-nitrosylation by NOS2 in the regulation of inflammatory mediators (9, 10).In the past, the p50 monomer was felt to be the probable target for NOS2-mediated S-nitrosylation of the p50-p65 heterodimer. This rationale was based on the initial identification of a single redox-sensitive cysteine (Cys-62) located in the DNA-binding region of p50 (11). Interestingly, this cysteine ...

No abstract

Background Post-translational modification of proteins by S-nitrosylation serves as a major mode of signaling in mammalian cells and a growing body of evidence has shown that transcription factors and their activating pathways are primary targets. S-nitrosylation directly modifies a number of transcription factors, including NF-κB, HIF-1, and AP-1. In addition, S-nitrosylation can indirectly regulate gene transcription by modulating other cell signaling pathways, in particular JNK kinase and ras. Scope of review The evolution of S-nitrosylation as a signaling mechanism in the regulation of gene transcription, physiological advantages of protein S-nitrosylation in the control of gene transcription, and discussion of the many transcriptional proteins modulated by S-nitrosylation is summarized. Major conclusions S-nitrosylation plays a crucial role in the control of mammalian gene transcription with numerous transcription factors regulated by this modification. Many of these proteins serve as immunomodulators, and inducible nitric oxide synthase (iNOS) is regarded as a principal mediatiator of NO-dependent S-nitrosylation. However, additional targets within the nucleus (e.g. histone deacetylases) and alternative mechanisms of S-nitrosylation (e.g. GAPDH-mediated trans-nitrosylation) are thought to play a role in NOS-dependent transcriptional regulation. General significance Derangement of SNO-regulated gene transcription is an important factor in a variety of pathological conditions including neoplasia and sepsis. A better understanding of protein S-nitrosylation as it relates to gene transcription and the physiological mechanisms behind this process is likely to lead to novel therapies for these disorders. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.It has become increasingly appreciated that protein S-nitrosylation, the covalent attachment of a nitroso group to a cysteine thiol side chain, is a principle mechanism by which nitric oxide modulates numerous cellular functions and phenotypes (1, 2). These include G-protein-coupled receptor signaling (3-5), death receptor-mediated apoptosis (6 -9), vesicular trafficking (10 -12), stimulation of prostaglandin synthesis (13-15), hypoxia-dependent control of blood flow (16 -18), and the unfolded protein response (19). In addition, aberrant S-nitrosylation is implicated in pathologies such as tumor initiation and growth (20, 21), neurodegeneration (19,22,23), and pulmonary hypertension (24).The three isoforms of nitric-oxide synthase (neuronal NOS/ NOS1, iNOS/NOS2, 2 and endothelial NOS/NOS3) are well established mediators of S-nitrosylation, and numerous studies have demonstrated that their localization is critical for S-nitrosylation of target proteins. For example, binding of iNOS to COX2 is required for S-nitrosylation and activation of prostaglandin synthesis (13), whereas the subcellular localization of endothelial NOS is a major determinant of S-nitrosylation-mediated protein trafficking (25). Moreover, although it is generally assumed that S-nitrosylation reactions are nonenzymatic, there is precedence for hemoglobin-dependent S-nitrosylation of the anion exchanger protein AE1 (26) and ceruloplasmin-dependent S-nitrosylation of glypican-1 (27) and GSH (28), consistent with a recent report that metalloproteins may play a general role in SNO synthesis (29).By contrast with progress in elucidating the enzymatic determinants of S-nitrosylation, the intracellular mediators of denitrosylation and their possible contributions to overall Snitrosylation status have only recently gained attention. By analogy to phosphorylation (where kinases and phosphatases together regulate phosphorylation), the steady-state level of S-nitrosylation is the net difference between nitrosylation and denitros...