Nitrosylation. the prototypic redox-based signaling mechanism.
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Allosteric Regulation↗
Biomedical subjects
J S Stamler
Publications and source records attributed to J S Stamler.
Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO.
We have shown previously that at physiologically relevant oxygen tension (pO(2) approximately 10 mmHg), NO S-nitrosylates 1 of approximately 50 free cysteines per ryanodine receptor 1 (RyR1) subunit and transduces a calcium-sensitizing effect on the channel by means of calmodulin (CaM). It has been suggested that cysteine-3635 is part of a CaM-binding domain, and its reactivity is attenuated by CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999) J. Biol. Chem. 274, 36831-36834]. Therefore, we tested the hypothesis that the effect of NO was mediated by C3635. The full-length RyR1 single-site C3635A mutant was generated and expressed in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO modulation of channel activity and reduced S-nitrosylation by NO to background levels but did not affect NO-independent channel modulation by CaM or the redox sensitivity of the channel to O(2) and glutathione. Our results reveal that different cysteines within the channel have been adapted to serve in nitrosative and oxidative responses, and that S-nitrosylation of the cysteine-containing CaM-binding domain underlies the mechanism of CaM-dependent regulation of RyR1 by NO.
Amino Acid Substitution↗
S-N dissociation energies of S-nitrosothiols: on the origins of nitrosothiol decomposition rates.
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Biological Transport, Active↗
Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen.
We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O(2) with nitric oxide (NO) to form nitrate [HMP-Fe(II)O(2) + NO --> HMP-Fe(III) + NO(3)(-)] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO(-) equivalent (followed by the formation of N(2)O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O(2) concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO(2) because the enzyme is then in the relatively inactive nitrosyl form [k(off)/k(on) for NO (0.000008 microM) k(off)/k(on) for O(2) (0.012 microM) and K(M) for O(2) = 30-100 microM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O(2)/NO concentrations. We find that, at biologically relevant O(2) concentrations, HMP preferentially binds NO (not O(2)), which it then reacts with oxygen to form nitrate (in essence NO(-) + O(2) --> NO(3)(-)). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.
Bacterial Proteins↗
Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.
Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.
Adenosine Diphosphate↗
S-nitrosothiol repletion by an inhaled gas regulates pulmonary function.
NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O(2)-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O(2)/O(2)(-). This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O(2) or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.
Administration, Inhalation↗
A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans.
Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.
Aldehyde Oxidoreductases↗
Classes of thiols that influence the activity of the skeletal muscle calcium release channel.
The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.
Animals↗
Inhibition of NF-kappa B by S-nitrosylation.
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.
Active Transport, Cell Nucleus↗
Export by red blood cells of nitric oxide bioactivity.
Previous studies support a model in which the physiological O2 gradient is transduced by haemoglobin into the coordinate release from red blood cells of O2 and nitric oxide (NO)-derived vasoactivity to optimize oxygen delivery in the arterial periphery. But whereas both O2 and NO diffuse into red blood cells, only O2 can diffuse out. Thus, for the dilation of blood vessels by red blood cells, there must be a mechanism to export NO-related vasoactivity, and current models of NO-mediated intercellular communication should be revised. Here we show that in human erythrocytes haemoglobin-derived S-nitrosothiol (SNO), generated from imported NO, is associated predominantly with the red blood cell membrane, and principally with cysteine residues in the haemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Interaction with AE1 promotes the deoxygenated structure in SNO-haemoglobin, which subserves NO group transfer to the membrane. Furthermore, we show that vasodilatory activity is released from this membrane precinct by deoxygenation. Thus, the oxygen-regulated cellular mechanism that couples the synthesis and export of haemoglobin-derived NO bioactivity operates, at least in part, through formation of AE1-SNO at the membrane-cytosol interface.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
S-nitrosylation: spectrum and specificity.
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Amino Acid Motifs↗
Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat.
This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.
Animals↗
Physiology of nitric oxide in skeletal muscle.
In the past five years, skeletal muscle has emerged as a paradigm of "nitric oxide" (NO) function and redox-related signaling in biology. All major nitric oxide synthase (NOS) isoforms, including a muscle-specific splice variant of neuronal-type (n) NOS, are expressed in skeletal muscles of all mammals. Expression and localization of NOS isoforms are dependent on age and developmental stage, innervation and activity, history of exposure to cytokines and growth factors, and muscle fiber type and species. nNOS in particular may show a fast-twitch muscle predominance. Muscle NOS localization and activity are regulated by a number of protein-protein interactions and co- and/or posttranslational modifications. Subcellular compartmentalization of the NOSs enables distinct functions that are mediated by increases in cGMP and by S-nitrosylation of proteins such as the ryanodine receptor-calcium release channel. Skeletal muscle functions regulated by NO or related molecules include force production (excitation-contraction coupling), autoregulation of blood flow, myocyte differentiation, respiration, and glucose homeostasis. These studies provide new insights into fundamental aspects of muscle physiology, cell biology, ion channel physiology, calcium homeostasis, signal transduction, and the biochemistry of redox-related systems.
Animals↗
Nitrosative stress: protection by glutathione-dependent formaldehyde dehydrogenase.
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Aldehyde Oxidoreductases↗
The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions.
Ion channels have been studied extensively in ambient O2 tension (pO2), whereas tissue PO2 is much lower. The skeletal muscle calcium release channel/ryanodine receptor (RyR1) is one prominent example. Here we report that PO2 dynamically controls the redox state of 6-8 out of 50 thiols in each RyR1 subunit and thereby tunes the response to NO. At physiological pO2, nanomolar NO activates the channel by S-nitrosylating a single cysteine residue. Among sarcoplasmic reticulum proteins, S-nitrosylation is specific to RyR1 and its effect on the channel is calmodulin dependent. Neither activation nor S-nitrosylation of the channel occurs at ambient PO2. The demonstration that channel cysteine residues subserve coupled O2 sensor and NO regulatory functions and that these operate through the prototypic allosteric effector calmodulin may have general implications for the regulation of redox-related systems.
Animals↗
Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin.
S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.
Allosteric Regulation↗
Protection from nitrosative stress by yeast flavohemoglobin.
Yeast hemoglobin was discovered close to half a century ago, but its function has remained unknown. Herein, we report that this flavohemoglobin protects Saccharomyces cerevisiae from nitrosative stress. Deletion of the flavohemoglobin gene (YHB1) abolished the nitric oxide (NO)-consuming activity of yeast cells. Levels of protein nitrosylation were more than 10-fold higher in yhb1 mutant yeast than in isogenic wild-type cells after incubation with NO donors. Growth of mutant cells was inhibited by a nitrosative challenge that had little effect on wild-type cells, whereas the resistance of mutant cells to oxidative stress was unimpaired. Protection conferred by yeast flavohemoglobin against NO and S-nitrosothiols was seen under both anaerobic and aerobic conditions, consistent with a primary function in NO detoxification. A phylogenetic analysis indicated that protection from nitrosative stress is likely to be a conserved function among microorganismal flavohemoglobins. Flavohemoglobin is therefore a potential target for antimicrobial therapy.
Aerobiosis↗
An apoptotic model for nitrosative stress.
Nitric oxide overproduction has been implicated in the pathogenesis of many disorders, including artherosclerosis, neurodegenerative diseases, inflammatory and autoimmune diseases, and cancer. The common view holds that nitric oxide-induced cellular injury is caused by oxidative stress. This theory predicts that interactions between reactive nitrogen species and reactive oxygen species produce powerful oxidants that initiate cell death programs. Cytokine-treated murine macrophages are the prototype of this form of cellular injury. Here we report that generation of reactive nitrogen species upon lipopolysacharide/interferon-gamma stimulation of RAW 264.7 cells is largely divorced from production of reactive oxygen species, and that oxidative stress is not principally responsible for cell death (in this model). Rather, the death program is induced mainly by a nitrosative challenge, characterized by the accrual of nitrosylated proteins without a major alteration in cellular redox state. Moreover, interactions between reactive oxygen and nitrogen species may alter the balance between pathways that yield nitrite and nitrate, without impacting the level of S-nitrosylation or extent of cell death. Our results thus (1) provide new insights into NO-related metabolic pathways, (2) demonstrate that apoptotic injury can be caused by nitrosative mechanisms, and (3) establish a model for nitrosative stress in mammalian cells.
2-Naphthylamine↗