Biological significance of nitric oxide-mediated protein modifications

Andrew J. Gow, Christiana R. Farkouh, David A. Munson, Michael A. Posencheg, and Harry Ischiropoulos

Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104


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Nitric oxide (NO), despite an apparently simple diatomic structure, has a wide variety of functions in both physiology and pathology and within every major organ system. It has become an increasingly important scientific challenge to decipher how this wide range of activity is achieved. To this end a number of investigators have begun to explore how NO-mediated posttranslational modifications of proteins may represent mechanisms of cellular signaling. These modifications include: 1) binding to metal centers; 2) nitrosylation of thiol and amine groups; 3) nitration of tyrosine, tryptophan, amine, carboxylic acid, and phenylalanine groups; and 4) oxidation of thiols (both cysteine and methionine residues) and tyrosine. However, two particular modifications have recently received much attention, nitrosylation of thiols to produce S-nitrosothiol and nitration of tyrosine residues to produce nitrotyrosine. It is the purpose of this review to examine the possibility that these modifications may play a role in NO-mediated signaling.

posttranslational modification; S-nitrosothiol; nitrotyrosine; nitrosylation; nitration



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In order for a protein modification to be a physiologically relevant signal it must fulfill certain criteria, namely: 1) the modification must be specific, 2) it must be reversible and preferentially via an enzyme-controlled mechanism, 3) its formation must occur on a physiological time scale, often via an enzyme-catalyzed reaction, and 4) depending on the target, the modification should result in either activation or inhibition. Clearly the classic signaling modification of phosphorylation fulfills these criteria, as it occurs at specific consensus sequences, its formation is catalyzed by kinases, and its removal is facilitated by phosphatases. Protein phosphorylation can result in either protein activation or inhibition. How do the known protein modifications mediated by nitric oxide (NO) relate to these criteria?


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One of the principal metalloproteins that interacts with NO is soluble guanylate cyclase (sGC). sGC is a heme-containing protein found in the cytosol of many cell types. It exists as a heterodimer composed of {alpha}- and {beta}-subunits. The NH2 terminus of each of these subunits contains a heme-binding domain. It is through this heme moiety that NO binds to sGC (38). The iron center of the heme moiety is bound to the enzyme via a histidine residue in the {beta}1-subunit (86). When NO binds to the ferrous state of the heme group [oxidation of the heme iron to the ferric state results in enzyme inactivation and heme loss (39)], it severs the bond between the heme iron and the histidine residue via an axial dislocation of the iron. This results in a conformational change that activates the enzyme (15), resulting in the production of cyclic GMP and the initiation of downstream signaling processes, such as vasorelaxation. Recently, it has been suggested that NO activation of sGC may be more complex and may involve a second site, although the precise nature and mechanism of this alternative binding site are unclear (8, 9).

The deactivation of sGC is also somewhat controversial. Two hypotheses for the mechanism exist. First, it is possible that NO simply dissociates from the heme group, which results in a reversal of the activating conformational change in the heme pocket. Second, it is also possible that there may be some extrinsic factor, such as glutathione, that could interact with the NO-iron bond, resulting in a more rapid dissociation than would otherwise occur (11). However, it is clear that the binding of NO with sGC results in a NO-protein interaction that is specific and reversible, occurs on a physiological time scale, and results in the activation of a biological process.

Although the interaction between NO and sGC results in activation of the enzyme and subsequent physiological processes, the interaction of NO with cytochrome c oxidase results in an inhibitory process. Cytochrome c oxidase is the terminal electron acceptor in the respiratory chain of mitochondria and many bacteria. It catalyzes the oxidization of cytochrome c and the concomitant conversion of oxygen to water, coupling this process to the export of a proton against the electrochemical gradient. The enzyme contains three metal centers involved in redox reactions, which take place during enzyme function. These centers include: a copper-copper dimer (CuA), heme A, and a binuclear heme-copper coupled center (heme a3/CuB) (91). NO binds to the reduced form of cytochrome c oxidase at the binuclear heme-copper center as a competitive inhibitor of oxygen, with dissociation rates similar to its binding of sGC. This is the classic physiological interaction of NO with cytochrome c oxidase (48). However, when the enzyme is oxidized, NO can also bind at the copper moiety of the binuclear center instead of the iron moiety. This may be more likely to occur at higher oxygen tensions when cytochrome c oxidase is predominantly oxidized. Under these conditions NO reacts with bound oxygen to produce nitrate and inactivate the enzyme (14). In either scenario, NO controls oxygen consumption and inhibits ATP production via inhibition of cellular respiration (34).

NO is also capable of interacting with nonheme metal centers, such as metallothionein (MT), which results in the liberation of intracellular zinc. This cysteine-rich protein is an avid binder of divalent cations in general and of zinc specifically. Recently, it has been demonstrated that NO binding to MT results in dissociation of zinc from the thiolate clusters, increasing intracellular zinc levels and potentially inhibiting apoptosis (78). This may have many physiological effects, as zinc is incorporated into a number of proteins as a structural component, including enzymes and transcription factors. It has also been proposed that MT-released zinc may inhibit mitochondrial respiration (90). Whatever effects the released zinc may have, this signaling mechanism represents a method by which a redox-active molecule such as NO can have effects on the intracellular homeostasis of an entirely redox-insensitive molecule, zinc (27). MT is not the only metallothiolate protein that can be regulated by NO. Aconitase, a critical enzyme in the Krebs cycle, contains a 4Fe-4S cluster and has been shown to be inhibited by NO-related molecules, although the mechanism is unclear (13, 16, 36).

Therefore, it would appear that within the context of metal signaling, NO can interact with a variety of centers to produce both activation (sGC and zinc release) and inhibition (cytochrome c oxidase and aconitase) that is reversible and occurs on a physiological timescale. Specificity is conferred by the nature of the metal centers themselves.


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As a result of its redox capacity, NO is able to nitrosate a number of nucleophilic sites on proteins, including amines, aromatic rings, alcohols, and reduced sulfur (thiol). Although all of these modifications have been of much historical interest, as far as the toxicological effects of exposure to nitrogen oxides is concerned, it is only recently that such modification has been considered to be of physiological relevance. The modification of thiol residues by NO to produce a S-nitrosothiol (SNO) was originally proposed to have a role in physiology by Stamler and colleagues (79). Since then, a wide variety of proteins have been found to be susceptible to S-nitrosylation. A recent review showed that 115 different proteins have so far been identified as targets for SNO formation (80). These proteins cover a wide range of functions, including kinases, channels, transcription factors, structural proteins, proteases, and respiratory enzymes. Therefore, it seems possible that S-nitrosylation may represent a physiological signaling pathway for NO, and as such we should consider whether it fits the criteria of a classic signaling pathway.

There are three separate pathways by which SNO can be formed: 1) via the formation of a higher oxide of nitrogen through autooxidation of NO, 2) via direct reaction followed by electron abstraction, or 3) via catalysis at metal centers. These pathways are summarized in Fig. 1. Classically, the formation of SNO has been considered to occur via the formation of a nitrosating intermediate such as N2O3 following the autooxidation of NO (49, 88). Critically important to such a reaction pathway is the requirement for two NO molecules to generate the nitrosating intermediate. This requirement makes such a reaction highly dependent on the local concentration of NO within a system. As such it will occur more readily under inflammatory conditions where the flux of NO is drastically increased and also under hydrophobic conditions where NO's lipophilicity causes it to accumulate. This is in contrast to both metal-catalyzed synthesis of SNO and direct reaction, which has been proposed to occurs via an RSNOH radical intermediate (28). Both of these mechanisms are first order with respect to NO and hence could occur more readily under physiological concentrations of NO seen in hydrophilic areas. These reactions both require the abstraction of an electron, either via the metal catalyst in conjunction with the nitrosylation of the thiol or following the formation of the RSNOH intermediate. It is unclear whether these reactions are favored by deprotonation of the thiol moiety, in which case they will occur more readily under hydrophilic conditions.



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Fig. 1. Pathways of S-nitrosylation. S-nitrosothiol (SNO) can be formed by interaction with 1) oxidants (such as molecular oxygen and superoxide) via the intermediacy of higher oxides of nitrogen (such as nitrogen dioxide, dinitrogen trioxide, and peroxynitrite), or 2) reduced thiol via the formation of an RSNOH radical intermediate, or 3) metal centers (such as copper and iron) either operating as catalysts or through the generation of nitrosonium cation.

 
Special consideration needs to be given to the understanding of metal-catalyzed SNO synthesis, as this can occur both nonspecifically and with the metal cofactors of certain proteins. Two vascular proteins have been shown to synthesize SNO via their metal center, namely hemoglobin (Hb) (31) and ceruloplasmin (40). Ceruloplasmin is a plasma protein with multiple copper-binding sites. Functionally, the role of ceruloplasmin had not been identified, although it has been implicated in the antioxidant capacity of plasma. Inoue and colleagues (40) have shown that ceruloplasmin can act as an SNO synthase and suggested that this may be a major physiological function for this protein. The ability of copper to catalyze both nitrosylation and denitrosylation of thiol has been established and appears to occur via a radical mechanism involving Cu(I) (20, 83). Hb provides an interesting model for protein-mediated SNO formation (30, 31, 73), in that the thiol moiety, cysteine {beta}93, is moved during the structural shift from the relaxed (R) state to the tense (T) state. Within the R state, it is in close proximity (~9 ) to the heme iron and is fully protected from contact with the solvent surface of the protein. However, in the T state it is rotated ~120° and brought into contact with the solvent surface. Furthermore, it is placed in close contact with the salt bridge, which is critical to the control of the Bohr shift. This structural shift may explain the lack of stability of SNO within the T state (45). The reversible nitrosylation of Hb has been suggested to play a role in the control of vascular relaxation, and in this regard, the anion exchanger 1 (70) and {gamma}-glutamyl cysteine transpeptidase (58) have been implicated to play a role in SNO transport.

The previous discussion reveals a number of mechanisms by which SNO can be synthesized under physiological conditions; however, for this modification to operate as a signaling mechanism, it is necessary for some degree of specificity to be obtained. Examination of two of the known proteins that are modified by S-nitrosylation, the N-methyl-D-aspartate (NMDA) receptor and Hb, gives an indication of one potential mechanism for specificity. The NMDA receptor is a member of the glutamate receptor class, which includes the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainite receptors, all of which contain redox-active cysteines. Only the NMDA receptor forms SNO, and only this receptor is regulated by NO. All three receptors contain a cysteine at positions 744 and 798, but the NMDA receptor contains a third cysteine at position 399 within the NR2A subunit (50, 55). Within the NMDA receptor, the three cysteine residues are flanked by acidic and basic residues, whereas in the other receptors they are not. This pattern of acidic and basic flanking residues is also seen in Hb. Indeed, this positioning of acidic and basic residues has been proposed to promote nitrosylation reactions (81). It has been suggested that the strict requirement is for proton donating and receiving groups to be within close proximity of the S{gamma}-atom within the three-dimensional structure of the protein (2). Therefore, examinations of primary sequence can lead to both false negative and positive identifications of potential sites of nitrosylation. Recently, other proteins have been found to form SNO in vivo that do not have these flanking residues, even within the three-dimensional structure, such as caspases-3 and -9 (62, 63). It would appear that these proteins contain an alternative motif in which the cysteine residue is buried within a hydrophobic span of residues. Such a sequence could promote the formation of SNO either by direct reaction or by autooxidation as a result of the increased solubility of NO in hydrophobic areas (68).

So far, one has seen that S-nitrosylation can occur in biological systems at specific sites and that the reaction can be catalyzed by known proteins, namely ceruloplasmin and Hb (31, 40). But is the reaction reversible on a time scale appropriate for a signaling mechanism? A number of chemical mechanisms exist for the decay of SNO, namely, homolytic cleavage [potentially catalyzed by light or Cu(I)], heterolytic cleavage to produce nitrosonium ion, and oxidation to produce disulfide and nitroxyl anion (1). All of these reactions are certainly viable in biological systems; however, it would appear that there also exists the possibility of enzymatic degradation. The glutathione-dependent formaldehyde reductase, which is expressed from prokaryotes to mammals, was recently shown to possess SNO reductase activity (59). This opens the possibility that, just as the kinases serve to control the phosphorylation signal transduction pathway, there exists an enzymatic pathway that controls SNO signaling.

The final criterion required for a signaling mechanism is that the modification can produce either activation or inhibition depending on the target protein. Examination of channel proteins best shows that SNO formation can produce both alterations. A number of different channel proteins have been identified as being capable of forming SNO; however, two are regulated in vivo by S-nitrosylation, the NMDA and the ryanodine receptors (RyR). As mentioned previously the NMDA receptor is inhibited by SNO formation (50, 55), and this modification has been linked to the control of this signaling pathway. However, the RyR is specifically S-nitrosylated on a single cysteine residue (among 50 potentially modifiable moieties), and this modification results in an increase in the channel opening probability, i.e., activation (82). Therefore, it would appear that S-nitrosylation fits the criteria of a signaling mechanism.


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Protein tyrosine nitration is a covalent protein modification resulting from the addition of a nitro (–NO2) group onto one of the two equivalent ortho carbons of the aromatic ring of tyrosine residues. Although the production of nitrotyrosine residues has commonly been used as a marker of pathological disease processes and of oxidative stress (42), evidence that the process of nitration meets the criteria for a physiologically relevant signal has been provided. Protein tyrosine nitration appears to be catalyzed, primarily by metalloproteins. An abundance of evidence has indicated that myeloperoxidase, eosinophil peroxidase, myoglobin, and the cytochrome P-450s catalyze the oxidation of nitrite to nitrogen dioxide, which is capable of nitrating tyrosine residues (12, 85, 89). Moreover, myeloperoxidase also catalyzes protein nitration by peroxynitrite, the product of the near diffusion-limited reaction of NO with superoxide (22). Metalloproteins such as Mn superoxide dismutase (61) and prostacyclin synthase (74) could catalyze their own nitration from peroxynitrite. Nonenzymatic sources of tyrosine nitration include the intermediate of the reaction between peroxynitrite with carbon dioxide (26, 56, 60) and the acidification of nitrite to form nitrous acid, an agent capable of nitrating tyrosine residues (51).

Both metalloprotein-catalyzed and nonenzymatic nitration processes are sufficiently rapid and can certainly occur on the physiological time scale. How about the specificity of the process? Certainly, the enzymatically catalyzed nitration of proteins is expected to confer specificity, as either metalloproteins would catalyze their own nitration or the nitration of proteins within a short radius of their location. Proximity-facilitated selectivity has been demonstrated in a cellular model of myeloperoxidase transcytosis, where nitrated fibronectin has been spatially associated with myeloperoxidase (7). The advent of proteomic approaches, as well as the development of numerous immunological and analytical methodologies, has revealed that protein nitration is limited to specific proteins (6, 25, 84). Although the proximity to the generating system is responsible in part for this selectivity, the in vivo nitration of soluble and abundantly expressed proteins such as actin (3) and {alpha}-synuclein (24) suggests the existence of additional biochemical and biophysical reasons for selective protein nitration. We have hypothesized that, analogous to other modifications of tyrosine residues in proteins such as phosphorylation and sulfation, nitration may also follow similar sequence and structural requirements (43).

Specific amino acid sequences serve as mechanisms for specificity in other tyrosine-mediated signaling systems. For example, tyrosine kinases phosphorylate tyrosine residues in the presence of a specific peptide sequence [Lys or Arg]-X-X-X-[Asp or Glu]-X-X-X-Tyr (47). It is possible that tyrosine could be a target of nitration if located within an analogous specific peptide sequence. However, to date there is little evidence supporting a unique sequence required for tyrosine residue nitration. A more likely mechanism for specificity of tyrosine nitration appears to be a consequence of local environment of tyrosine residues within the secondary and tertiary structure of the protein (analogous to the requirement for acidic and basic residues in the vicinity of cysteine for nitrosylation). Tyrosyl protein sulfotransferase provides a model for this kind of specificity, with stringent requirements for sulfation to occur (69). These include the presence of a nearby negative charge, usually in position 1, the presence of turn-inducing amino acids, and the absence of steric hindrances. Tyrosine residue nitration may also have similar structural requirements with the presence in close proximity of a negative charge is one consistent feature in most known sites of tyrosine nitration. However, the significance of this association is unknown, and ongoing site-directed mutational analysis in model proteins (57) is expected to improve our understanding. Therefore, it would appear that, despite an inability to define all the criteria required to be a target for tyrosine nitration, this process occurs at a particular subset of tyrosine residues in selective proteins and thus can be said to be specific.

Is there any evidence for reversibility within the nitrative process? Evidence that nitrated proteins could be removed by different cellular proteolytic systems has been provided (29, 41, 46, 67, 77). Recently, the dynamic nature of tyrosine nitration was demonstrated within mitochondria (5, 18). Moreover, a putative specific activity, termed denitrase, which removes the nitro group without degrading the protein, has been preliminarily shown to be present in tissues, but the identification of such activity awaits further confirmation. Alternatively, nitrated proteins may be removed via immunological processes. The persistence of nitrated proteins under certain conditions elicits an antigenic response. Specific monoclonal antibodies that recognize only the nitrated form but not the unmodified {alpha}-synuclein have been produced, suggesting that nitrated peptides can be differentially recognized and processed by the immune system (24). Recently, Birnboim and coworkers (10) demonstrated that nitrated peptides elicit a robust immune response in transgenic mice incapable of mounting a response to the native peptide. These findings suggested that protein nitration might induce immune responses to autologous proteins and profoundly influence immunological responses in autoimmune and inflammatory diseases.

Overall, protein tyrosine nitration appears to be a specific process that can occur through a variety of mechanisms upon a physiologically relevant time scale. Although there is no clearly defined mechanism of removal of this modification, there is evidence that such a signal could be "turned off" by either protein degradation or denitration.


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In the preceding sections we have attempted to outline potential biochemical routes for NO to control cellular signaling events. Are there any known examples of such signaling, in general, and within the lung, in particular? One of the most well-developed models of NO signaling via posttranslational modification of proteins occurs within the Ras family of GTPases (53). This is a relatively large family of proteins in which a particular subset contains a cysteine residue at position 118. Interestingly, within these proteins the cysteine is flanked by an acidic and a basic residue (a sequence that has been suggested to promote SNO formation), and these proteins are nitrosylated in vivo (21, 44). Furthermore, SNO formation activates these signaling proteins by destabilizing bound GDP and promoting nucleotide exchange (53, 87). Ras proteins that lack this cysteine are insensitive to NO; in addition, mutation of this residue removes the NO sensitivity of p21Ras (52, 64). The precise mechanism of Ras nitrosylation is not entirely clear, although the preponderance of the evidence indicates a nucleophilic attack followed by electron abstraction (87). The RyR provides another example of a well-defined NO-mediated posttranslational modification that is involved in signaling (19). The RyR contains 84 redox-active cysteine residues; however, nitrosylation of one of these residues activates the receptor, resulting in an increased channel opening probability (82). Furthermore, mutation of a single residue abolishes NO sensitivity of the receptor. The RyR is critical to Ca2+ handling within skeletal and cardiac muscle cells and thus plays a critical role in controlling diaphragm contractility and may play a role in NO-mediated control of diaphragm function (71).

The importance of appropriate NO metabolism within the lung has been demonstrated in a variety of human diseases, including asthma (17, 23), acute respiratory distress syndrome (76), and cystic fibrosis (32). Indeed, the use of SNO has been proposed as a therapeutic modality within the lung (72), and SNO repletion by inhalation of O-nitrosoethanol has been shown to modulate pulmonary arterial pressures (65, 66). In an animal model of pulmonary inflammation, surfactant protein D deletion, NO metabolism is disrupted with a reduction in the formation of SNO and an increase in nitration within the lung tissue (4). The importance of the interaction between surfactant proteins and NO metabolism has been highlighted by the studies performed with surfactant protein A. Surfactant protein A is readily nitrated by exposure to peroxynitrite (33) or activated alveolar macrophages (92), and such modified protein is unable to aggregate lipids or bind Pneumocystis carinii (35, 93). Upon exposure to pathophysiological levels of reactive nitrogen species, surfactant protein A is specifically nitrated at tyrosines 166 and 164. Interestingly, the addition of the nitration catalyst carbon dioxide results in nitration of a further tyrosine at position 161 (92). The dynamic nature of the relationship between surfactant proteins and NO metabolism is demonstrated in the mycoplasmicidal activity of alveolar macrophages, which appears to occur through a surfactant protein A-mediated production of peroxynitrite (37).


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From the considerations given in this review, one can appreciate that NO can produce different protein modifications, namely nitrosylation of metal and thiol centers and nitration of tyrosine residues, each with their own specific signaling consequences. A note of caution should be considered when examining the literature on this topic, as measurement of these modifications is technique sensitive, and a variety of approaches is required to confirm pathophysiological relevancy. The type of modification produced will be dependent on the concentration of potential targets, the flux of NO, the environment, and the presence of cofactors. It seems probable that this chemical variety provides the key to understanding the complexity of cellular signals that can be produced by NO. Furthermore, it is clear that the when and where of NO production are critical in determining its signaling consequences, which may explain why NO synthase localization is so precisely controlled (75).

It is worth noting that the three modifications discussed here vary in their chemical complexity. Nitrosylation of metal centers is a simple binding relationship and thus involves no transfer of electrons and can occur at a low energy cost. The formation of SNO requires the removal of a single electron, i.e., the conversion of the nitrogen from an oxidation state of 2 to 3. Therefore, this is a more energetically costly reaction and is thus more energetically costly to perform. Nitration of tyrosine residues represents a significant oxidation of the nitrogen from 2 to 5 and thus energetically requires the most input both for formation and decomposition. It is tempting to suggest that this energetic variation allows for a temporal shift in NO signaling, i.e., the greater the energetic cost of the signal the lower the frequency of the modulation. Thus high-frequency NO signals would be mediated by nitrosylation (such as vasorelaxation), whereas low-frequency signals (such as protein turnover and immune responses) would be performed via nitration. What is clear is that NO signaling can occur through a variety of chemical modifications that should be considered when examining how NO interacts within a biological system.


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Address for reprint requests and other correspondence: H. Ischiropoulos and A. J. Gow, Children's Hospital of Philadelphia, Abramson Research Center, Rm. 416, 34th & Civic Center Blvd., Philadelphia, PA 19104 (E-mail: ischirop{at}mail.med.upenn.edu and Gow{at}email.chop.edu)


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