Nitric oxide signaling gone awry: Nitration of glutamine synthetase and hyperammonemia in sepsis
Authors: Aimee Landar, Victor M. Darley‐Usmar
Journal: Hepatology (2005)

Under physiological conditions, nitric oxide (NO)-dependent signaling pathways function through different mechanisms involving the interaction with metal centers and the modification of proteins through amino acid residues. For example, the well-known interaction of soluble guanylate cyclase with NO involves reversible binding to the ferrous heme of the enzyme and increased synthesis of cGMP through this allosteric regulation.1 By analogy with multiple examples of the reversible and controlled activation of receptors by second messengers, this interaction of NO can be classified as a "classical" receptor-mediated signaling pathway.2 Recently, a similar heme nitrosylation reaction has also been identified in the mitochondrion with the terminal member of the electron transport chain cytochrome c oxidase.3-5 Binding of NO to this enzyme results in the reversible inhibition of mitochondrial respiration, the increased reduction of distal members of the respiratory chain and, as a consequence, increased formation of superoxide which is then converted by the enzyme MnSOD to hydrogen peroxide.6, 7 It is these conditions which result in the posttranslational modification of proteins including S-nitrosation and tyrosine nitration. The current debate in the area of redox biology is centered on determining to what extent the posttranslational modifications contribute to cell signaling or the mechanisms of disease. NO, nitric oxide; ROS, reactive oxygen species; RNS, reactive nitrogen species; GS, glutamine synthetase. In this respect, mitochondria are particularly interesting since they are both a source and target of reactive oxygen and nitrogen species (ROS/RNS). Initially, ROS formation from the mitochondrion was regarded as symptomatic of bioenergetic dysfunction and therefore deleterious. This perspective is now changing with the recognition that diffusion of hydrogen peroxide from the organelle results in the activation of specific redox cell signaling pathways.7-10 While many details of these mechanisms remain to be defined, the loss of control of this signaling pathway is likely to be associated with a pathological response. For example, we have proposed that high levels of NO lead to a bioenergetic dysfunction with some of the characteristics of hypoxia, but with enhanced ROS formation and the oxidative modification of mitochondrial proteins; a process we have termed NO-dependent hypoxia or nitroxia.9 Furthermore, in the presence of high levels of NO the ability of MnSOD to convert superoxide to hydrogen peroxide can be exceeded and result in the formation of peroxynitrite (Fig. 1). An additional amplification of this process may also arise from the nitration and inactivation of MnSOD leading to enhanced peroxynitrite levels as has been reported in chronic inflammatory diseases.11 At a molecular level, the formation of peroxynitrite will switch NO-dependent modification from reversible heme nitrosylation reactions to irreversible modification of proteins through oxidative and nitrative pathways.12, 13 The switch between nitrosative and nitrative NO-dependent modification of proteins in the mitochondrion. A simplified representation of the respiratory chain is shown with NO binding to cytochrome c oxidase through a nitrosylation reaction at the heme group in the oxygen binding site. This leads to increased hydrogen peroxide formation, shown here from complex III but complex I is also a potential site. Under nonpathological conditions superoxide is converted to hydrogen peroxide that can leave the mitochondrion and participate in redox cell signaling. Excess NO as occurs in sepsis or decreased MnSOD activity switches the nitrosative to nitrative reactions that can lead to the modification of tyrosine residues as described in this editorial. In the current issue of HEPATOLOGY, the study by Görg et al. is an interesting example of the pathological consequences of switching NO signaling from nitrosative to nitrative pathways.14 This study is important because it links a precise molecular target for protein nitration to the pathophysiology associated with high levels of NO under inflammatory conditions. Understanding the role of NO in sepsis has been a long-term goal of many investigators in the redox signaling field. In this and similar pathological conditions, the increased production of NO from the inducible NO synthase is associated with increased ROS.15, 16 In this context, a primary culprit for the switch from physiological cell signaling to pathology in sepsis is peroxynitrite.17 However, the formation of nitrotyrosine has become a widely accepted marker of nitrative biology through additional mechanisms beyond peroxynitrite including myeloperoxidase and its reaction with nitrite.18 Over the past decade, it has been a challenge to identify precise molecular targets for nitrative reactions that could lead to cellular dysfunction. Perhaps surprisingly, nitration reactions are not extensive, even under pathological conditions, and relatively few specific targets have been defined to date. For example, nitrotyrosine formation has been shown to be associated with loss of function in certain enzymes such as superoxide dismutase, prostacyclin synthase, and cytochrome c.2, 12, 18, 19 One of the problems in this area of research has been to link posttranslational modification of proteins, such as nitration, to physiological and pathological outcomes. This study is exceptional in this regard since it identifies a precise molecular target that is modified by nitration, and further defines the connection between this modification and development of hyperammonemia. This condition occurs in response to sepsis, and the authors report that it is due to the tyrosine nitration and subsequent inactivation of the enzyme glutamine synthetase (GS). The authors demonstrate that LPS induces protein tyrosine nitration after 24 hours in rat liver that follows increased iNOS expression. GS, which is a major enzyme involved in the scavenging of ammonia from the blood, was highly nitrated 24 hours post LPS treatment, and this nitration was accompanied by a loss of GS enzymatic activity. The nitrotyrosine residue in the active site of purified GS was modified. In the perfused livers this was associated with elevated ammonia release and decreased glutamine synthesis, indicating a loss of GS function. Interestingly, after immunodepletion with the nitrotyrosine antibody, there is a loss of approximately 50% of GS protein consistent with the conclusion that it is highly nitrated in vivo. The concept advanced in this paper is that the stoichiometric nitration of a protein such as GS leads to a proportional change in enzymatic function. This is certainly borne out by the data presented here, but may not be applicable to all nitration targets. It is well recognized that proteins with related function are structurally organized in regulated metabolomes or signaling domains that are architecturally arranged on a specific scaffold. The amino acid residues that form the essential basis of these oligomeric structures, when modified, may lead to failure to form either metabolic or signaling domains. Through such a mechanism, the cooperativity inherent in the formation of such intracellular structures will result in greater dysfunction beyond a stoichiometric loss of enzyme activity. The persistent emergence of key targets for nitration associated with cytoskeletal proteins and enzymes involved in both glycolysis and in the mitochondrion again supports the importance of domain structures in channeling nitration reactions.19-26 In addition, there are a number of potential explanations for these findings, which are certainly not mutually exclusive including: (1) susceptibility of the target tyrosine to nitration, (2) proximity to the location of the nitrating agent, (3) differential activity of the putative denitrase enzymes, (4) bias in the detection methods using affinity approaches, and (5) bias due to the differential representation of a subproteome within a given cell. However, the fact that nitration of GS has such a profound effect on ammonia concentrations in the blood is remarkable and indicates how nitrative modifications can impact on physiological and pathological responses, relevant to sepsis. Naturally, this study also raises a number of questions related to the basic molecular mechanisms of nitration in inflammation. Biologically, this could lead to failure through loss of the regulation of metabolism in entire metabolomic structures. In this model, cumulative hits of 10% or less in distinct elements in a metabolic or signaling domain could lead to altered function and pathology. This paradigm integrates the key concepts of systems biology, redox signaling and the pathology of oxidative and nitrative stress. The definition of the nitro-proteome, in response to conditions such as sepsis are giving us the first glimpses of how such modifications may contribute to human pathophysiology and this important study describes one such functional consequence. The authors are grateful to the National Institutes of Health for research support (NIH AA13395 to VDU).