Inactivation Of Glutamine Synthetase Research Articles

Glutamine Metabolism Is Altered in Myeloproliferative Neoplasms and Represents a Potential Novel Therapeutic Target

Cancer cells exhibit metabolic reprogramming to ensure sufficient production of macromolecules used to promote cell growth and proliferation. Previous studies have shown that proliferating cancer cells rely significantly on glutamine metabolism as a source of nitrogen and carbon to facilitate nucleotide and lipid biosynthesis, respectively, and to generate metabolites and/or maintain redox homeostasis. Despite this phenomenon being well characterized in many different solid and blood cancer types, very few studies have investigated the metabolic landscape of myeloproliferative neoplasms (MPNs). Glutamine catabolism begins via the uptake of glutamine through sodium-dependent transporters such as SLC1A5, and its transport to the mitochondria where it then undergoes deamination by glutaminase (GLS1) to produce glutamate. This conversion by GLS1 is the rate limiting step in the central glutaminolysis axis, making it a preferential point of study and therapeutic targeting. Indeed, many studies have explored the glutaminolysis pathway in a variety of cancers to decipher potential targets for treatment, but these studies have been extremely limited in MPNs and the potential mechanisms with respect to MPN driver mutations (CALR, JAK2, MPL) and their differential utilization of glutamine have yet to be fully explored. One previous study demonstrated that in JAK2V617F mutant-Ba/F3 cells, GLS1 expression was significantly increased and that GLS1 inhibition served as a promising intervention to suppress colony formation in patient samples but not healthy donors. To more comprehensively decipher the role of glutamine metabolism in MPNs driven by all three driver mutations ( CALR, JAK2, and MPL), we performed RNA-sequencing analysis on peripheral blood mononuclear cells (PMBCs) from MPN patients and found that genes involved in glutamine metabolism are significantly altered across the board. In contrast to the previous study mentioned above, our data demonstrate that GLS1 is not significantly up-regulated, and that MPN PBMCs are not sensitive to GLS1 inhibition. Rather, we characterized another key enzyme in glutamine metabolism, glutamine synthetase (GS) as a molecular and metabolic dependency in MPNs. GS catalyzes the reversion of glutamate into glutamine and serves to regulate nitrogen metabolism, support nucleotide biosynthesis, and most importantly synthesize glutamine to satiate the demand caused by the cancer cells' affinity for high glutamine levels. We found that GS is significantly up-regulated at the mRNA, protein, and activity level in MPNs driven by all three driver mutations, and that genetic and pharmacological inactivation of GS abrogates MPN cell proliferation in vitro. In summary, we have demonstrated that GLS1 may not be an effective therapeutic target for MPNs, and instead characterize GS as a novel dependency and potential point of therapeutic intervention.

Oxidative Inactivation of Human Glutamine Synthetase: Defining the Role of Tyrosine Nitration

Oxidative Inactivation of Human Glutamine Synthetase: Defining the Role of Tyrosine Nitration

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) invitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Mechanisms of Oxidative Inactivation of Human Glutamine Synthetase

Mechanisms of Oxidative Inactivation of Human Glutamine Synthetase

Macrophage-derived glutamine boosts satellite cells and muscle regeneration

Muscle regeneration is sustained by infiltrating macrophages and consequent satellite cell (SC) activation1–4. Macrophages and SC communicate in different ways1–5 but their metabolic interplay was never investigated so far. Here, we found that muscle injuries and aging are characterized by intratissutal glutamine restriction. Low glutamine levels endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity at the expense of glutamate dehydrogenase-1 (GLUD1)-mediated glutamine oxidation. Glud1 knockout (KO) macrophages display constitutively high GS activity which prevents glutamine shortage. Import of macrophage-derived glutamine by SC through the glutamine-transporter SLC1A5 activates mTOR and promotes SC proliferation and differentiation. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischemia, or aging. Conversely, SLC1A5 blockade in SC or GS inactivation in macrophages negatively affects SC functions and muscle regeneration. These results highlight a metabolic cross-talk between SC and macrophages whereby macrophage-derived glutamine sustains SC functions. Thus, GLUD1 targeting offers new therapeutic opportunities for the regeneration of injured or aged muscles.

Peroxynitrite-mediated Oxidation and Inactivation of Human Glutamine Synthetase

Peroxynitrite-mediated Oxidation and Inactivation of Human Glutamine Synthetase

Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.

A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria.

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5′ UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

OP-38 - Insights into the mechanisms of peroxynitrite-mediated inactivation of human glutamine synthetase

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the CNS, it is mainly located in the cytosol of astrocytes, playing an important role in detoxification of ammonia and excess glutamate. Alterations in GS activity and oxidative posttranslational modifications have been found in vivo. Herein, we studied the effect of oxidants on recombinant hGS function and structure with a combination of experimental and computational studies. Peroxynitrite caused a dose-dependent inactivation of hGS and 3-nitrotyrosine (NT) formation. In addition, higher molecular weight aggregates were formed likely due to 3,3´-dityrosine crosslinks. pH studies suggested that 3,3´-dityrosine formation had a larger impact on GS activity than nitration. MS analysis identified some critical residues modified, namely Tyr 185 and 269. H 2 O 2 -dependent thiol oxidation did not impact on GS activity. Exposure of human astrocytes to SIN-1 led to GS nitration and decreased enzyme activity. Molecular dynamic studies in GS (Issoglio et al. Biochemistry 2016) are being performed in nitrated forms of the enzyme to define with an atomic level of detail how tyrosine oxidative modifications affect GS structure and activity.

2 - Oxidative Inactivation of Human Glutamine Synthetase: Biochemical and Computational Studies

Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. In the central nervous system, it is mainly located in the cytosol of astrocytes, playing an important role in ammonia detoxification and prevention of glutamate-dependent excitotoxicity. Alterations in GS activity may lead to astroglial dysfunction, affecting neuronal function and survival. In its native state, GS has a decameric structure, with a monomer molecular weight of 42 KDa. Several in vitro and in vivo studies in plants, bacteria and mammals, have shown that GS activity is highly susceptible to biologically-relevant reactive oxygen species, in particular peroxynitrite (ONOO–). Peroxynitrite-derived radicals promote tyrosine nitration yielding 3-nitrotyrosine (3-NT). Tyrosine nitration of GS has been identified as one of the main oxidative modifications associated to enzyme inactivation in pathological conditions; however, the critical residues involved and the molecular mechanisms participating in GS inactivation are still undefined. Herein we have worked with human glutamine synthetase (hGS), which was expressed in E. coli and further purified to homogeneity. Then, we studied the effect of different oxidants on hGS function and structure, paying special attention to the oxidative mechanisms of inactivation, by combining classical biochemical assays with molecular dynamic simulations. Peroxynitrite addition caused a dose-dependent inactivation of hGS, associated to an increase in 3-NT levels. In addition, we were able to detect the formation of high molecular weight aggregates, resistant to the action of reductants, probably due to di-tyrosine crosslinks. Both, tyrosine nitration and aggregate-formation strongly correlated with enzyme inactivation, however, pH-dependent studies suggested that di-tyrosine formation had a larger impact on enzyme activity, compared to nitration. Under these conditions we also observed thiol-oxidation and disulfide formation, although these modification did not impact on enzyme activity. In addition, peptide-mapping MS analysis identified critical modified residues, namely Tyr 185 and 269. In parallel to the experimental studies, molecular dynamic simulations were performed to understand the catalytic mechanisms of the reaction with the goal of defining, with an atomic level of detail, how the oxidative postranslational modifications affect activity, in particular tyrosine residues associated to the substrate binding sites.

Over-expression of human clusterin increases stress resistance and extends lifespan in Drosophila melanogaster

Clusterin is a disulfide-linked heterodimeric glycoprotein that has been implicated in a variety of biological processes. Its expression has been shown to be elevated during cellular senescence and normal aging, but it is uncertain whether clusterin protects against aging or whether its expression is a consequence of aging. To investigate the functions of clusterin during organismal aging, we established transgenic Drosophila alleles to induce the expression of the secretory form of human clusterin (hCluS) using the Gal4/UAS system. hCluS protein (∼60kDa) was detected in both adult homogenates and larval hemolymphs of flies ubiquitously overexpressing hCluS (da-Gal4>UAS-hCluS) and in motoneurons (D42-Gal4>UAS-hCluS). Interestingly, the mean lifespans of these hCluS-overexpressing flies were significantly greater than those of control flies that exhibited no hCluS induction. hCluS-overexpressing flies also showed significantly greater tolerance to heat shock, wet starvation, and oxidative stress. Furthermore, amounts of reactive oxygen species (ROS) in whole bodies were significantly lower in hCluS-overexpressing flies. In addition, clusterin was found to prevent the inactivation of glutamine synthetase (GS) by metal-catalyzed oxidation (MCO) in vitro, and this protection was only supported by thiol-reducing equivalents, such as, DTT or GSH, and not by ascorbate (a non-thiol MCO system). Furthermore, this protection against GS inactivation by clusterin was abolished by reacting clusterin with N-ethylmaleimide, a sulfhydryl group-modifying agent. Taken together, these results suggest that a disulfide-linked form of clusterin functions as an antioxidant protein via its cysteine sulfhydryl groups to reduce ROS levels and delay the organismal aging in fruit flies.

Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: critical role of arginine amino acid residues for glutamine synthetase inactivation

The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.

Inactivation of astrocytic glutamine synthetase by hydrogen peroxide requires iron

The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H 2O 2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H 2O 2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H 2O 2 (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H 2O 2, iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H 2O 2, with approximately 37% of activity remaining 3 h after incubation with 1 mM H 2O 2. This inactivation was prevented by the iron chelators 2,2′-dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H 2O 2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.

Posttranscriptional Regulation of Glutamine Synthetase in the Filamentous Cyanobacterium Anabaena sp. PCC 7120: Differential Expression between Vegetative Cells and Heterocysts

Genes homologous to those implicated in glutamine synthetase (GS) regulation by protein-protein interaction in the cyanobacterium Synechocystis sp. strain PCC 6803 are conserved in several cyanobacterial sequenced genomes. We investigated this GS regulatory mechanism in Anabaena sp. strain PCC 7120. In this strain the system operates with only one GS inactivation factor (inactivation factor 7A [IF7A]), encoded by open reading frame (ORF) asl2329 (gifA). Following addition of ammonium, expression of gifA is derepressed, leading to the synthesis of IF7A, and consequently, GS is inactivated. Upon ammonium removal, the GS activity returns to the initial level and IF7A becomes undetectable. The global nitrogen control protein NtcA binds to the gifA promoter. Constitutive high expression levels of gifA were found in an Anabaena ntcA mutant (CSE2), indicating a repressor role for NtcA. In vitro studies demonstrate that Anabaena GS is not inactivated by Synechocystis IFs (IF7 and IF17), indicating the specificity of the system. We constructed an Anabaena strain expressing a second inactivating factor, containing the amino-terminal part of IF17 from Synechocystis fused to IF7A. GS inactivation in this strain is more effective than that in the wild type (WT) and resembles that observed in Synechocystis. Finally we found differential expression of the IF system between heterocysts and vegetative cells of Anabaena.

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen

Reversible male sterility and doubled haploid plant production are two valuable technologies in F(1)-hybrid breeding. F(1)-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F(1)-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.

Analysis of methionine oxides and nitrogen-transporting amino acids in chilled and acclimated maize seedlings

In maize seedlings, chilling causes a reduction of glutamine synthetase (GS) activity, while acclimation protects GS (manuscript submitted). Since ROS can oxidize both protein-bound and free Met to methionine sulfoxide (MSO) and further to methionine sulfone (MSO2, a GS inhibitor), it was hypothesized that the chilling-induced oxidative stress may cause accumulation of MSO and MSO2, thus contributing to the inactivation of GS. MSO2 preferentially inhibited the chloroplastic isoform, GS2. HPLC analysis of polar amino acids from coleoptiles + leaves, mesocotyls and roots of control, chilled, acclimated, acclimated and chilled and chilled and rewarmed plants revealed that free MSO and MSO2 do not accumulate after low temperature treatments. Nevertheless, acclimation significantly increased the expression of putative protein methionine sulfoxide reductase (PMSR), especially in mesocotyls. Different low temperature treatments caused complex changes in the profiles of N-transporting amino acids, Asp, Glu, Asn and Gln.

A theoretical steady state analysis indicates that induction of Escherichia coli glnALG operon can display all-or-none behavior

The nitrogen starvation response in Escherichia coli is characterized by the enhanced expression of Ntr regulon, comprising hundreds of genes including the one coding for nitrogen-assimilating glutamine synthetase (GS) enzyme. The biosynthesis and activity of GS is regulated mainly by nitrogen and carbon levels in the cell and monitored by three functionally separable interconnected modules. Here, we present the steady-state modular analysis of this intricate network made up of a GS bicyclic closed-loop cascade, a NRII-NRI two-component system, and an autoregulated glnALG operon encoding genes for GS, NRII, and NRI. Our simulation results indicate that the transcriptional output of glnALG operon is discrete and switch-like, whereas the activation of transcription factor NRI is graded, and the inactivation of GS is moderately ultrasensitive to input stimulus glutamine. The autoregulation of the NRII-NRI two-component system was found to be essential for the all-or-none induction of the glnALG operon. Furthermore, we show that the autoregulated two-component system modulates the total active GS by delineating the GS activity from its biosynthetic regulation. Our analysis indicates that the exclusive relationship between GS activity and its synthesis is brought about by the autoregulated two-component system. The modularity of the network endows the system to respond differently to nitrogen depending on the carbon status of the cell. Through a system-level quantification, we conclude that the discrete switch-like transcriptional response of the E. coli glnALG operon to nutrient starvation prevents the premature initiation of transcription and may represent the desperate attempt by the cell to survive in limiting conditions.

Glutamine synthetase degradation is controlled by oxidative proteolysis in the marine cyanobacterium Prochlorococcus marinus strain PCC 9511

Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe 3+/NADH/NADH oxidase/O 2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.

Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress

Glutamine synthetase (GS) is an astrocytic enzyme that is essential for the glutamate-glutamine cycle between neurons and astrocytes. To measure the effects of oxidative stress on the activity of GS in astrocytes, astrocyte-rich primary cultures from the brains of wild-type and glutathione peroxidase-1 deficient mice (GPx1(-/-)) were exposed to a chronic hydrogen peroxide-generating system consisting of xanthine oxidase, hypoxanthine and superoxide dismutase. The specific activity of GS was strongly diminished by chronic exposure to hydrogen peroxide in astrocytes cultured from both mouse lines. After 60 min of oxidative stress in the presence of 5 mU/mL, 10 mU/mL and 20 mU/mL of xanthine oxidase, the specific GS activity of wild-type astrocytes was reduced to 47%, 22% and 13% of the initial activity, respectively. For all activities of xanthine oxidase applied, astrocytes from GPx1(-/-) mice experienced a significantly greater rate of GS inactivation compared to their wild-type counterparts. These results confirm that GS is sensitive to inactivation by chronic peroxide stress in viable astrocytes and show that glutathione peroxidase-1 helps to protect GS from inactivation by oxidative stress.

Lipopolysaccharide-induced tyrosine nitration and inactivation of hepatic glutamine synthetase in the rat

Glutamine synthetase (GS) in the liver is restricted to a small perivenous hepatocyte population and plays an important role in the scavenging of ammonia that has escaped the periportal urea-synthesizing compartment. We examined the effect of a single intraperitoneal injection of lipopolysaccharide (LPS) in vivo on glutamine synthesis in rat liver. LPS injection induced expression of inducible nitric oxide synthase, which was maximal after 6 to 12 hours but returned toward control levels within 24 hours. Twenty-four hours after LPS injection, an approximately fivefold increase in tyrosine-nitrated proteins in liver was found, and GS protein expression was decreased by approximately 20%, whereas GS activity was lowered by 40% to 50%. GS was found to be tyrosine-nitrated in response to LPS, and immunodepletion of tyrosine-nitrated proteins decreased GS protein by approximately 50% but had no effect on GS activity. Together with the finding via mass spectrometry that peroxynitrite-induced inactivation of purified GS is associated with nitration of the active site tyrosine residue, our data suggest that tyrosine nitration critically contributes to inactivation of the enzyme. In line with GS inactivation, glutamine synthesis from ammonia (0.3 mmol/L) in perfused livers from 24-hour LPS-treated rats was decreased by approximately 50%, whereas urea synthesis was not significantly affected. In conclusion, LPS impairs hepatic ammonia detoxification by both downregulation of GS and its inactivation because of tyrosine nitration. The resulting defect of perivenous scavenger cell function with regard to ammonia elimination may contribute to sepsis-induced development of hyperammonemia in patients who have cirrhosis.