Nitrosylation
2001; Cell Press; Volume: 106; Issue: 6 Linguagem: Inglês
10.1016/s0092-8674(01)00495-0
ISSN1097-4172
AutoresJonathan S. Stamler, Santiago Lamas, Ferric C. Fang,
Tópico(s)Ammonia Synthesis and Nitrogen Reduction
ResumoTwo signaling systems, based on the principle of posttranslational modification of proteins, are conserved throughout evolution and influence most aspects of cellular physiology—one is phosphorylation and the other is redox based. Both exemplify dynamic regulation of protein function by reversible modification, and they govern many of the same signal transduction pathways through overlapping sets of cellular targets. They are also prone to malfunction in human disease. However, while many basic principles of signal transduction have emerged from studies of phosphorylation, the redox-based mechanism has remained far more enigmatic. A major difficulty has been to comprehend how specificity of action is achieved. Recent advances in understanding how nitric oxide (NO) regulates protein function are now providing answers. At the recent Juan March Foundation Workshop on “Regulation of Protein Function by Nitric Oxide (Nitrosylation and Nitrosative Stress),” several core principles emerged. First, NO groups modify cysteine thiols and transition metal centers of a broad functional spectrum of proteins, and with remarkable spatial and temporal resolution (for over 100 representative examples, see Table S1 in Supplementary Material available online at http://www.cell.com/cgi/content/full/106/6/675/DC1 ). Second, the majority of these proteins are regulated by S-nitrosylation of a single critical cysteine residue within an acid-base or hydrophobic structural motif, which may also be subject to oxygen- or glutathione-dependent modification. Thus, S-nitrosylation emerges as a prototypic redox-based signal. Third, the same NO-related posttranslational modifications that operate as specific signals in mammalian cells can be used to fight invasion by microbes and cancer cells. That is, nitrosylation can disrupt the function of critical proteins in pathologically proliferating cells in what is referred to as nitrosative stress. This theme will be dealt with in greater detail in the latter part of this report, which focuses on antimicrobial actions of NO and the cellular defense mechanisms that protect specifically against NO-related species, as well as on the deleterious consequences of redox-based modification of proteins. Allosteric proteins control and coordinate signaling pathways in all living cells. Allosteric theory holds that posttranslational modifications of proteins operate by shifting a dynamic equilibrium between inactive and active conformations (Monod et al. 1965Monod J. Wyman J. Changeux J.-P. On the nature of allosteric transitions a plausible model.J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (5900) Google Scholar). Phosphorylation in particular has served as a model of how protein modification triggers conformational changes that regulate signal transduction. These fundamental concepts of allosteric regulation in fact originate in studies of the posttranslational modification of hemoglobin by small diatomic ligands. Thus, whereas phosphorylation clearly lies at the heart of many signal transduction pathways, the principles of protein regulation by posttranslational modification—first formalized by Monod, Wyman, and Changeux—are better established for NO/O2. This hemoglobin-based paradigm for protein regulation (J.S.S., Duke University, Durham, NC) has been expanded recently by the discovery of an enzymatic function for hemoglobin, operating within the confines of a membrane-localized signaling module by binding to its target protein and introducing NO groups (much as a kinase introduces phosphates) (Pawloski et al. 2001Pawloski J.R. Hess D.T. Stamler J.S. Export by red blood cells of nitric oxide bioactivity.Nature. 2001; 409: 622-626Crossref PubMed Scopus (467) Google Scholar). In this case, a redox-based signal that is initiated through an O2-induced allosteric transition in hemoglobin is propagated by S-nitrosylation of an anion exchanger to subserve intercellular communication. Two key themes have emerged from these studies of hemoglobin: (1) the importance of allostery in posttranslational modification of proteins by NO, and (2) the role of O2/redox as an allosteric effector. The ryanodine receptor/calcium release channel (RyR), which is controlled by O2-regulated and calcium-calmodulin (CaM)-linked S-nitrosylation, serves to further illustrate these general principles. Biochemical analyses have shown that O2 concentration dynamically controls the redox state of 6–8 thiols per RyR subunit and thereby regulates S-nitrosylation of a single channel thiol (Eu et al. 2000bEu J.P. Sun J. Xu L. Stamler J.S. Meissner G. The skeletal muscle calcium release channel coupled O2 sensor and NO signaling functions.Cell. 2000; 102 (b): 499-509Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). These data can be interpreted (J.S.S. and G. Meissner, University of North Carolina at Chapel Hill, NC), by analogy to hemoglobin, in terms of an equilibrium between two alternative structures that is governed by O2 tension. In this model, a redox-driven conformational change in the channel provides a hydrophobic compartment that concentrates NO and O2, thus generating nitrosylating equivalents. The observation that S-nitrosylation activates the RyR only when it is complexed by CaM is explained by the location of the critical cysteine within a 1-5-10 CaM binding motif. It was proposed that calcium binding to RyR-resident apo-calmodulin unmasks this critical thiol, allowing S-nitrosylation to produce a function-regulating perturbation of the CaM/RyR complex. It is important to emphasize both the precision with which NO acts on the skeletal muscle RyR and the intricate nature of NO regulation. Nitric oxide S-nitrosylates only 1 out of 50 free cysteines per RyR subunit, but this is sufficient to alter the CaM/RyR interaction and sensitize the channel to both positive and negative regulation by calcium (Sun et al. 2001Sun J. Xin C. Eu J.P. Stamler J.S. Meissner G. Cysteine 3635 is responsible for skeletal muscle ryanodine receptor modulation by NO.Proc. Natl. Acad. Sci. USA. 2001; in pressGoogle Scholar). Furthermore, this mechanism only operates at a restricted, physiological O2 concentration. Oxygen can therefore be viewed as producing a preparatory redox-based modification of the channel, which serves to facilitate S-nitrosylation. Such precisely regulated NO/O2-mediated posttranslational modifications of proteins would have been difficult to envision even one year ago. In recent years, the notion that the transduction of signals relies on the free diffusion of molecules within the cell has been replaced by an appreciation that signaling takes place within the confines of subcellular compartments that are critical for both specificity of targeting and propagation of signals (Davare et al. 2001Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2.Science. 2001; 293: 98-101Crossref PubMed Scopus (427) Google Scholar). That is, the enzymes that effect posttranslational modifications, the second messenger systems, and the effector molecules that propagate the signals are discretely colocalized so that signals can be optimally generated, processed, and channeled. Substrates are effectively delivered to these multiprotein complexes by anchoring proteins, and so-called scaffolding proteins provide structural integrity as well as influence the composition of the complex. Progress in the area of calcium signaling in particular has revealed that temporal as well as spatial resolution is required for efficient signal transduction. One immediately appreciates the resulting conundrum: while NO gained prominence as a diffusible signaling molecule with a promiscuous sphere of influence, thus creating a new paradigm in cellular communication, the field of signal transduction increasingly emphasized compartmentalization as a core determinant of specificity. Further complicating matters, NO biologists focused almost exclusively on a second messenger, cGMP (the product of soluble guanylate cyclase), which mediates most of its effects through a single protein kinase. Thus NO, a molecule identified with more important and disparate biological functions than any other, would rely on only a single kinase effector, when the human genome is predicted to contain over a thousand kinases (Hunter 2000Hunter T. Signaling—2000 and beyond.Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2187) Google Scholar), each linked to a specific and limited set of responses. The current picture of NO biology is, of course, very different: G kinase is only a small part of the story (Jaffrey et al. 2001Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Protein S-nitrosylation a physiological signal for neuronal nitric oxide.Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1184) Google Scholar, Lane et al. 2001Lane, P., Hao, G., and Gross, S.S. (2001). S-nitrosylation is emerging as a specific and fundamental posttranslational protein modification: head-to-head comparison with O-phosphorylation. Science's STKE, 1–9.Google Scholar), and the presentation by Doris Koesling (Rurh-Universitat, Bochum, Germany) emphasized that it is likely to be identified with a restricted and highly specific set of effects. Koesling reported that one isoform of “soluble” guanylate cyclase is, in fact, membrane associated, through PDZ-domain interactions with the scaffolding protein PSD95. This localization serves to place the cyclase in the immediate proximity of a neuronal isoform of nitric oxide synthase (NOS), within the confines of a signaling complex. Immunohistochemical images of cGMP associated with the plasma membrane (Kobzik et al. 1994Kobzik L. Reid M.B. Bredt D.S. Stamler J.S. Nitric oxide in skeletal muscle.Nature. 1994; 372: 546-548Crossref PubMed Scopus (825) Google Scholar) raise the further possibility that the second messenger need not diffuse far to find a discrete and limited set of targets. We have learned that cells may contain multiple isoforms and splice variants of NOS. Discrete localization within subcellular compartments enables these enzymes to carry out different functions. For example, both skeletal and cardiac muscle coexpress three different isoforms of NOS in as many as four different locations: a plasmalemmal enzyme that regulates force production and blood flow, a mitochondrial NOS controlling respiration at the level of cytochrome c oxidase (the state of the art was discussed by Salvador Moncada, University College, London, UK), a sarcoplasmic reticular NOS involved in calcium homeostasis, and additional constitutive and inducible cytosolic isoforms of NOS whose exact location and function is unknown. Grigori Enikolopov (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) further described an important aspect of NO signaling by multiple forms of NOS. In PC12 cells, the three major NOS isoforms are sequentially upregulated during the switch from a proliferative to a differentiated phenotype. Enikolopov has used a high-throughput genomic approach to search for NO-regulated genes involved in cell proliferation and cell cycle control, and found many, including Rb and p53. Carl Nathan (Weill Medical College, Cornell University, Ithaca, NY) emphasized the potential of NO generated by even one enzyme to regulate many and disparate cellular responses. Based on differential expression patterns in wild-type and iNOS knockout macrophages, Nathan, Sabine Ehrt, and colleagues concluded that iNOS comediates the regulation of hundreds of genes by interferon-γ and Mycobacterium tuberculosis. It is known that the effects of iNOS on both host and microbial cells are largely cGMP-independent, as are the effects of NO on skeletal muscle and the majority of targets identified by Enikolopov. It will therefore be important to obtain a more complete picture of the substrates and partners of the various NOSs and of their exact location within mammalian cells, as well as in microbes and multicellular pathogens that contain a NOS. It has been almost a decade since it was discovered that the NMDA receptor (NMDAR) is regulated by S-nitrosylation, thus demonstrating that NO signaling can originate in or at the plasma membrane (Lipton et al. 1993Lipton S.A. Choi Y.B. Pan Z.H. Lei S.Z. Chen H.S. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.Nature. 1993; 364: 626-632Crossref PubMed Scopus (2240) Google Scholar). These studies presaged the identification of a multiple-component signaling module that now provides the best illustration of the spatiotemporal aspect of NO signaling (Figure 1). In this module, the neuronal isoform of NOS is brought into close proximity with the NMDAR by their mutual interactions with a scaffolding protein PSD95. This juxtaposition provides a localized Ca2+ stimulus for NOS, and NO bioactivity feeds back to control the activity of the channel. Downregulation of the NMDAR is mediated by S-nitrosylation of critical cysteines within the NR2a and NR1 regulatory subunits (Choi et al. 2000Choi Y.B. Tenneti L. Le D.A. Ortiz J. Bai G. Chen H.S. Lipton S.A. Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.Nat. Neurosci. 2000; 3: 15-21Crossref PubMed Scopus (361) Google Scholar), which were recently found to be nitrosylated constitutively in the brain (Jaffrey et al. 2001Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Protein S-nitrosylation a physiological signal for neuronal nitric oxide.Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1184) Google Scholar). nNOS is also coupled via the anchoring protein CAPON to the small G protein Dexras, which it activates by S-nitrosylation (Fang et al. 2000Fang M. Jaffrey S.R. Sawa A. Ye K. Luo X. Snyder S.H. Dexras1 a G protein specifically coupled to neuronal nitric oxide synthase via CAPON.Neuron. 2000; 28: 183-193Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) in much the same way that anchoring proteins colocalize kinases and their substrates. p21 ras, which is regulated by S-nitrosylation independently of Dexras, is also found within this NMDAR complex (Yun et al. 1998Yun H.Y. Gonzalez-Zulueta M. Dawson V.L. Dawson T.M. Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras.Proc. Natl. Acad. Sci. USA. 1998; 95: 5773-5778Crossref PubMed Scopus (174) Google Scholar), and a membrane-bound guanylate cyclase (vida supra) that is recruited by PSD95 has recently been added to the mix. It remains to be determined how NMDA-generated NO signals can be alternatively processed through two different small G proteins and guanylate cyclase. The discrete subcellular localization of NOSs within the confines of protein modules is necessary but not sufficient to achieve signaling specificity. This can be understood by appreciating that membrane signaling domains such as caveolae are estimated to contain as many as 50–100 proteins (Hunter 2000Hunter T. Signaling—2000 and beyond.Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2187) Google Scholar) and at least as many free thiols, but most are probably not sites of S-nitrosylation. Moreover, both transmembrane signaling systems (e.g., receptor tyrosine kinases), and intracellular signaling pathways (e.g., MAP kinase and JAK/STAT) that transmit signals from the plasma membrane to the nucleus, are regulated by NO at multiple loci that are not known to be closely associated with NOSs. Examples of recently identified targets in these categories that were covered at the symposium include EDGF receptor tyrosine kinase (Antonio Villalobo, University Autonoma de Madrid, Spain), JNK kinase (principle work by Choi and colleagues, Korea University, Seoul, South Korea), JAK kinase (Tyk2, Christian Bogdan, Friedrich-Alexander Universitat Erlangen, Germany), NF-κB (J.S.S., Duke University, Durham, NC for his group and others), and AP-1 (S.L., CSIC, Madrid, Spain). These kinases and transcription factors exemplify the many classes of NO targets that do not contain transition metals (with which NO can interact directly) and which are not G kinase (cGMP)-regulated (see Table S1 in Supplementary Material available online at http://www.cell.com/cgi/content/full/106/6/675/DC1). Rather, NO responsiveness is conferred by the presence of critical cysteine residues that have signature features (Hess et al. 2001Hess D.T. Matsumoto A. Nudelman R. Stamler J.S. S-nitrosylation spectrum and specificity.Nat. Cell Biol. 2001; 3: E46-E49Crossref PubMed Scopus (210) Google Scholar, Stamler et al. 1997bStamler J.S. Toone E.J. Lipton S.A. Sucher N.J. S)NO signals translocation, regulation, and a consensus motif.Neuron. 1997; 18 (b): 691-696Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar). In one scenario, the target cysteine is located between an acidic and a basic amino acid, as revealed in either the primary or tertiary structure. This motif supports general acid/base chemistry of S-nitrosylation/denitrosylation reactions and may play additional roles that are specific to each case. Sharon Campbell (University of North Carolina at Chapel Hill, NC) highlighted these principles in structural and biochemical studies of ras protein. Remarkably, the NMR structure and activity of S-nitrosylated p21 ras differs only slightly from that of native protein. However, stimulation of guanine nucleotide dissociation from p21 ras was evident in a system competent in S-nitrosylation/denitrosylation reactions, suggesting that SNO turnover may be required to propagate this NO-regulated signal. A mechanism was provided in which S-nitrosylation interferes with crucial stabilizing interactions between the acidic aspartate (adjacent to the cysteine) and GDP, to produce a guanine-exchange-factor-like effect. The only other known structure of an S-nitrosylated protein is that of hemoglobin, in which the acid-base motif functions both to regulate the pK of the thiolate and to maintain the protein alternately in NO-responsive and -resistant conformations. Presentations by S.L. and Alfred Hausladen (Duke University, Durham, NC) emphasized that the acid-base motif may apply for other reversible redox-based modifications of cysteines that would regulate protein function: -SNO, -SOH, -S-S and -S-glutathione (Klatt and Lamas 2000Klatt P. Lamas S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (643) Google Scholar, Stamler and Hausladen 1998Stamler J.S. Hausladen A. Oxidative modifications in nitrosative stress.Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (242) Google Scholar) (Table S1, see URL above). Electrostatic interactions that stablilize −OH and −glutathione attachments in proteins were invoked by Hauslasen and S.L., respectively. It is somewhat suprising that analysis of primary amino acid sequence has proven to be predictive of sites of S-nitrosylation in so many well-characterized cases (e.g., NMDAR, ras protein, and hemoglobin), given the statistical probability that proximal acids and bases would arise frequently in three-dimensional structure, but the list of examples continues to grow. Marie-Christine Broillet (Université de Lausanne, Switzerland) reported that the acid-base motif correctly identified the single critical cysteine in the C-terminal cytoplasmic domain of the β-subunit of the cyclic nucleotide-gated channel that confers NO responsiveness. Whereas the channel contains 4 free thiols (2 of which are in the C-terminal domain), only the thiol conforming to the motif is necessary and sufficient for NO regulation. This analysis is a continuation of electrophysiological studies on a homo-β form of the channel, which was found to be NO-regulated but not gated by cGMP (Broillet and Firestein 1997Broillet M.C. Firestein S. Beta subunits of the olfactory cyclic nucleotide-gated channel form a nitric oxide activated Ca2+ channel.Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Broillet showed that the NO-regulated channel isoform colocalizes with nNOS in brain regions that are not involved in olfaction, implying novel functions. There are now several examples where the acid-base motif is revealed only in the tertiary or quaternary structure of the protein. Jose Mato (Universidad de Navarra, Spain) showed that analysis of the crystal structure of methionine adenosyl transferase I/III, which contains 10 free cysteines including an active site thiol, was required to pinpoint the critical cysteine target of S-nitrosylation. Site-directed mutagenesis of the juxtaposed acidic and basic residues, which are cryptic with regard to the primary sequence, abrogated NO responsiveness (Perez-Mato et al. 1999Perez-Mato I. Castro C. Ruiz F.A. Corrales F.J. Mato J.M. Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol.J. Biol. Chem. 1999; 274: 17075-17079Crossref PubMed Scopus (121) Google Scholar). Mato has made a convincing case that methylation reactions, which underlie regenerative responses of the liver, are regulated by S-nitrosylation of this rate-limiting enzyme. Steven Gross (Weill Medical College, Cornell University, Ithaca, NY) demonstrated that a single cysteine in arginosuccinate synthetase provides a locus for NO regulation of NO synthesis. By inhibiting this enzyme, NO prevents the recycling of citrulline to arginine, which is the principle source of NOS substrate in inflammatory settings. Gross suggested that this critical cysteine has evolved specifically as an NO sensor: it is conserved in all mammals, but absent from microorganisms that do not possess a recognizable NOS, and it is used to differentiate nitrosative from oxidative signals. Specifically, both H2O2 and S-nitrosothiols (SNOs) inhibit the enzyme, but the effect of H2O2 is retained after mutation of the critical cysteine, whereas the SNO effect is lost. The requirements for such selectivity are known to be provided by an acid-base motif (Perez-Mato et al. 1999Perez-Mato I. Castro C. Ruiz F.A. Corrales F.J. Mato J.M. Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol.J. Biol. Chem. 1999; 274: 17075-17079Crossref PubMed Scopus (121) Google Scholar). An S-nitrosylation motif was also identified within the recently solved three-dimentional structure of aquaporin 1 (Hess et al. 2001Hess D.T. Matsumoto A. Nudelman R. Stamler J.S. S-nitrosylation spectrum and specificity.Nat. Cell Biol. 2001; 3: E46-E49Crossref PubMed Scopus (210) Google Scholar), which Jean-Luc Balligand (Univesité Catholique de Louvain Medical School, Belgium) indicated is regulated by NO and related molecules. The demonstration that eNOS and aquaporin 1 are colocalized within caveolae suggests that NO responsiveness may be the physiological correlate of the sensitivity to mercurials that has long characterized this water channel. Finally, the structural and redox requirements of SNO-forming reaction channels within proteins were highlighted by David Singel (Montana State University, Bozeman, MT), who demonstrated that NO groups can transfer directly from oxidized transition metals to nearby cysteines within proteins. These studies were done with hemoglobin, but it is apparent that tunneling of NO through and between proteins may be a common phenomenon. The acid-base motif is likely to operate preferentially in hydrophilic milieux, and alternative mechanisms are needed to explain the specific modification by NO of certain cysteines found in hydrophobic environments (Hess et al. 2001Hess D.T. Matsumoto A. Nudelman R. Stamler J.S. S-nitrosylation spectrum and specificity.Nat. Cell Biol. 2001; 3: E46-E49Crossref PubMed Scopus (210) Google Scholar). Indeed, it was initially recognized that the reactive cysteine forming the site of NO binding in oxygenated hemoglobin is located within a hydrophobic pocket, and that S-nitrosylation by free NO is critically dependent on this protein configuration (Gow and Stamler 1998Gow A.J. Stamler J.S. Reactions between nitric oxide and haemoglobin under physiological conditions.Nature. 1998; 391: 169-173Crossref PubMed Scopus (502) Google Scholar, Stamler et al. 1997aStamler J.S. Jia L. Eu J.P. McMahon T.J. Demchenko I.T. Bonaventura J. Gernert K. Piantadosi C.A. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient.Science. 1997; 276 (a): 2034-2037Crossref PubMed Scopus (914) Google Scholar). It has also been reported that the cysteines involved in regulating the turnover of palmitate on the neuronal proteins GAP43 and SNAP25 reside within a hydrophobic membrane binding region (Hess et al. 1993Hess D.T. Patterson S.I. Smith D.S. Skene J.H. Neuronal growth cone collapse and inhibition of protein fatty acylation by nitric oxide.Nature. 1993; 366: 562-565Crossref PubMed Scopus (292) Google Scholar). Most recently it was found that the single cysteine conferring NO responsiveness in the skeletal RyR resides within a 1,5,10 CaM binding region in a pocket of high relative hydrophobicity (see above). Robin Rosenfeld (of the Getzoff Laboratory, Scripps Research Institute, La Jolla, CA) reported that a cysteine in NOS, which is particularly susceptible to S-nitrosylation, is found in a hydrophobic domain, raising the possibility that the site may serve in allosteric regulation of NO synthesis or in the egress of NO bioactivity. Nedospasov et al. 2000Nedospasov A. Rafikov R. Beda N. Nudler E. An autocatalytic mechanism of protein nitrosylation.Proc. Natl. Acad. Sci. USA. 2000; 97: 13543-13548Crossref PubMed Scopus (133) Google Scholar have shown that such hydrophobic compartments enable S-nitrosylation by concentrating the NO and O2 reactants that generate nitrosylating equivalents. Thus, cysteine-containing hydrophobic compartments in proteins, exemplified by the 1,5,10 CaM binding region within RyR, define a hydrophobic motif for S-nitrosylation (Hess et al. 2001Hess D.T. Matsumoto A. Nudelman R. Stamler J.S. S-nitrosylation spectrum and specificity.Nat. Cell Biol. 2001; 3: E46-E49Crossref PubMed Scopus (210) Google Scholar). It is anticipated that such hydrophobic motifs will be found frequently within intramembrane segments of proteins, and may be used in certain cytosolic proteins as a means to overcome competition for NO by ambient glutathione. It has been recognized that all mammalian cells contain low levels of nitrosylated proteins. Although constitutive nitrosylation was initially presumed to directly reflect basal NOS activity, the regulation of steady-state levels of nitrosylated proteins is proving to be far more complex. An emerging theme is that levels of nitrosylation are regulated not only by enzymes that add NO groups but also by enzymatic (as well as redox-based or allosteric effector) mechanisms that remove them (Lai et al. 2001Lai T.S. Hausladen A. Slaughter T.F. Eu J.P. Stamler J.S. Greenberg C.S. Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.Biochemistry. 2001; 40: 4904-4910Crossref PubMed Scopus (109) Google Scholar, Lane et al. 2001Lane, P., Hao, G., and Gross, S.S. (2001). S-nitrosylation is emerging as a specific and fundamental posttranslational protein modification: head-to-head comparison with O-phosphorylation. Science's STKE, 1–9.Google Scholar, Mannick et al. 1999Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Fas-induced caspase denitrosylation.Science. 1999; 284: 651-654Crossref PubMed Scopus (691) Google Scholar), analogous to regulation of phosphorylation by kinases and phosphatases. In human B and T cell lines, for example, background amounts of nitrosylation are largely unaffected by NOS inhibition. However, apoptotic stimuli such as Fas crosslinking result in denitrosylation of a subset of proteins. Joan Mannick (University of Massachusetts, Worcester, MA) showed that one of these is the mitochondrial caspase-3. In this case, denitrosylation is required to activate the enzyme, which is then released from mitochondria to initiate apoptosis. In contrast, it was found that cytochrome c is nitrosylated prior to its release from mitochondria. Nitrosylated cytochrome c increases caspase activation (in the presence of Apaf1) relative to native protein. Thus, an apoptotic stimulus produces coordinated nitrosylation and denitrosylation events, which operate in concert to activate the apoptotic pathway. The mechanism of denitrosylation in these studies is not known. However, recent work supports the proposal that specific enzymes can govern levels of nitrosylation. It has been demonstrated that yeast and mice deficient in alcohol dehydrogenase III show impaired metabolism of S-nitrosoglutathione and accumulate S-nitrosylated proteins (Liu et al. 2001Liu L. Hausladen A. Zeng M. Que L. Heitman J. Stamler J.S. A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans.Nature. 2001; 410: 490-494Crossref PubMed Scopus (714) Google Scholar). Mutant cells are thus sensitized to nitrosative stress. Mato showed that intracellular glutathione can also facilitate denitrosylation reactions, emphasizing the role of cellular redox state in control of redox-based protein modifications. But it is important to appreciate that many NO groups in proteins are not accessible to glutathione and are not influenced by redox changes at the cellular level, consistent with involvement of specific “redox enzymes.” It has been suggested, for example, that the thioredoxin system might play a rol
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