Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Metabolic fingerprinting reveals extensive consequences of GLS hyperactivity.

BACKGROUND: High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways. METHODS: We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS). RESULTS AND CONCLUSIONS: Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected. GENERAL SIGNIFICANCE: By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

GLS1 Mutant Mice Display Moderate Alterations of Hippocampal Glutamatergic Neurotransmission Associated with Specific Adaptive Behavioral Changes.

Significant alterations in glutamatergic neurotransmission have been reported in major depressive disorder (MDD) that could underlie psychiatric traits. Studies were mainly interested in synaptic dysfunction in the prefrontal cortex, a key structure involved in depressive-like behavior, however hippocampus has been shown to be important in MDD. As cognitive deficits such as hippocampus-memory process were observed in MDD, we investigated in a mild hypoglutamatergic model behaviors related to depression and memory, synaptic transmission parameters and glutamatergic state specifically in the hippocampus. We thus characterized these phenotypes in adult male mice partially depleted in glutaminase type 1 or GLS1 (GLS1 HET), the enzyme responsible for glutamate synthesis in neurons, that we previously characterized as displaying moderate lower levels of glutamate in brain. We showed that GLS1 mutant mice display AMPA-R-mediated response deficits after prolonged repetitive stimulation with electrophysiological recording and inability to sustain glutamate release by microdialysis experiments with no consequences on behavioral spatial learning performances. However, their ability to escape from unpleasant but repeated escapable condition was attenuated whereas they were more immobile in the unescapable situation in the FST during re-test. These results show that GLS1 mutant mice display moderate impairments of hippocampal glutamatergic neurotransmission and moderate changes in adaptive behaviors that have been shown to participate to the development of depressive-like state.

MicroRNA-200c exacerbates the ischemia/reperfusion injury of heart through targeting the glutaminase (GLS)-mediated glutamine metabolism.

OBJECTIVE: Cardiac ischemia and reperfusion, the common pathophysiological processes during cardiovascular surgery, are followed by oxidative stresses during the restoration of blood flow to the tissue, known as ischemia/reperfusion (IR) injury. microRNAs (miRNAs) are a group of endogenous, short and noncoding RNAs that post-transcriptionally repress their target mRNA expressions. Currently, the roles of microRNAs in the IR are still under investigated. This study will investigate the roles and mechanisms of miRNAs in the ischemia/reperfusion injury of the heart. MATERIALS AND METHODS: A rat myocardial ischemia-reperfusion injury model was established in this study. MiR-200c expression was measured by qRT-PCR. MiR-200c mimics was transfected into rat H9c2 cardiomyocytes to test the effects of miR-200c on the glutamine metabolism. The glutamine uptake, glutamine dehydrogenase activity, α-ketoglutarate, and glutaminase were assessed. RESULTS: Here, we show that endogenous miR-200c expression is stimulated by IR in rat heart. We observed miR-200c expressions were induced by H2O2 treatments in H9c2 rat cardiomyocytes. Overexpression of miR-200c increased the ROS levels under H2O2. Moreover, the glutamine metabolism is suppressed by IR in rat heart. We identified miR-200c directly targets the glutaminase (GLS) through complimentary binding to the 3'UTR reagent of GLS. We report either knockdown of GLS by siRNA or overexpression of miR-200c suppresses glutamine metabolism in H9c2 cardiomyocytes. Notably, the miR-200c inhibitor-pretreated rat heart exhibits improved heart function in IR. CONCLUSIONS: This study reports an important function of miR-200c in the regulation of glutamine metabolism during ischemia/reperfusion injury and will contribute to the development of new diagnostic and therapeutic interventions for the protection of IR.

Glutaminase C overexpression in the brain induces learning deficits, synaptic dysfunctions, and neuroinflammation in mice.

Glutaminolysis, a metabolic process that converts glutamine to glutamate, is particularly important for the central nervous system since glutamate is the major transmitter of excitatory synapses. Glutaminase is the mitochondrial enzyme that catalyzes the first step of glutaminolysis. Two genes encode at least four isoforms of glutaminase in humans. Gls1 gene encodes isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC) through alternative splicing, whereas Gls2 gene encodes liver-type glutaminase isoforms. KGA and GAC have been associated with several neurological diseases. However, it remains unclear whether changes in their expressions can directly cause brain abnormalities. Using a transgenic approach, we generated mice that overexpressed GAC in the brain. The resulting transgenic mice had severe impairments in spatial and fear learning compared with littermate controls. The learning deficits were consistent with diminished hippocampal long-term potentiation in the hippocampal slices of the GAC transgenic mice. Furthermore, we found increases in astrocyte and microglia markers, inflammatory factors, and a decrease in synapse marker synaptophysin, suggesting neuroinflammation and synaptic changes in the GAC transgenic mouse brains. In conclusion, these findings provide the first evidence that GAC overexpression in the brain has deleterious effects on learning and synaptic integrity in vivo.

Increased Expression of Glutaminase in Osteoblasts Promotes Macrophage Recruitment in Periapical Lesions.

INTRODUCTION: Recently, we have shown that tissue hypoxia stimulates the progression of periapical lesions by up-regulating glycolysis-dependent apoptosis of osteoblasts. Other facets of hypoxia-induced metabolic reprogramming in disease pathogenesis require further investigation. In this study, we examined the connection between hypoxia-augmented glutamine catabolism in osteoblasts and the development of periapical lesions. METHODS: Primary human osteoblasts were cultured under hypoxia. The expression of glutaminase 1 (GLS1) was examined using Western blot analysis. The production of glutamate was measured by colorimetric assay. Knockdown of GLS1 was performed with small interfering RNA technology. C-C motif chemokine ligand 2 (CCL2) secretion and chemotaxis of J774 macrophages were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced periapical lesions, the relations between disease progression and osteoblastic expression of GLS1 or macrophage recruitment were studied. RESULTS: Hypoxia enhanced GLS1 expression and subsequent glutamate production in osteoblasts. Glutamate induced chemoattraction of macrophages by osteoblasts through up-regulation of CCL2 synthesis. Hypoxia promoted CCL2 secretion and macrophage recruitment through augmentation of glutaminolysis. Knockdown of GLS1 abolished hypoxia-induced effects. In rat periapical lesions, progressive bone resorption was significantly related to elevated GLS1 expression in osteoblasts and increased macrophage recruitment. CONCLUSIONS: In addition to the rise in glycolytic activity, the progression of periapical lesions is also associated with enhanced glutamine catabolism in osteoblasts. GLS1 may be a potential therapeutic target in the management of periapical lesions.

The expression of CG9940 affects the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila.

The CG9940 gene, which encodes the NAD(+) synthase protein in Drosophila, is conserved in human, zebra fish, and mosquito. NAD(+) synthase is a homodimer, which catalyzes the final step in de novo nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, an amide transfer from either ammonia or glutamine to nicotinic acid adenine dinucleotide (NaAD). Both the CG9940 and exercise are closely relative to NAD(+) level, and NAD(+) plays important roles not only in energy metabolism and mitochondrial functions but also in aging. In our study, the expression of CG9940 was changed by UAS/GAL4 system in Drosophila. Flies were trained by a training device. Cardiac function was analyzed by M-mode traces, climbing index was measured through negative geotaxis assay, and lifespan was measured via lifespan assays. The important new findings from our present study included the following: (1) the expression of the CG9940 could affect cardiac function, mobility, and lifespan in Drosophila. Over-expression of the CG9940 gene had positive effects on Drosophila, such as enhanced aging cardiac output, reduced heart failure, delayed age-related mobility decline, and prolonged lifespan, but lower-expression of the CG9940 had negative effects on them. (2) Different expressions of the CG9940 resulted in different influences on the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Both normal-expression and over-expression of the CG9940 resulted in positive influences on the adaptation of cardiac functions, mobility, and lifespan to exercise in aging Drosophila such as exercise slowed age-related decline of cardiac function, mobility and extent of lifespan in these flies, while lower-expression of the CG9940 led to negative impacts on the adaptation of mobility and lifespan to exercise in Drosophila.

mTORC1-Dependent Metabolic Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells.

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment.

The mechanistic target of rapamycin (mTOR) is hyperactivated in many types of cancer, rendering it a compelling drug target; however, the impact of mTOR inhibition on metabolic reprogramming in cancer is incompletely understood. Here, by integrating metabolic and functional studies in glioblastoma multiforme (GBM) cell lines, preclinical models, and clinical samples, we demonstrate that the compensatory upregulation of glutamine metabolism promotes resistance to mTOR kinase inhibitors. Metabolomic studies in GBM cells revealed that glutaminase (GLS) and glutamate levels are elevated following mTOR kinase inhibitor treatment. Moreover, these mTOR inhibitor-dependent metabolic alterations were confirmed in a GBM xenograft model. Expression of GLS following mTOR inhibitor treatment promoted GBM survival in an α-ketoglutarate-dependent (αKG-dependent) manner. Combined genetic and/or pharmacological inhibition of mTOR kinase and GLS resulted in massive synergistic tumor cell death and growth inhibition in tumor-bearing mice. These results highlight a critical role for compensatory glutamine metabolism in promoting mTOR inhibitor resistance and suggest that rational combination therapy has the potential to suppress resistance.

Opposing roles of glutaminase isoforms in determining glioblastoma cell phenotype.

Glutamine (Gln) and glutamate (Glu) play pivotal roles in the malignant phenotype of brain tumors via multiple mechanisms. Glutaminase (GA, EC 3.5.1.2) metabolizes Gln to Glu and ammonia. Human GA isoforms are encoded by two genes: GLS gene codes for kidney-type isoforms, KGA and GAC, whereas GLS2 codes for liver-type isoforms, GAB and LGA. The expression pattern of both genes in different neoplastic cell lines and tissues implicated that the kidney-type isoforms are associated with cell proliferation, while the liver-type isoforms dominate in, and contribute to the phenotype of quiescent cells. GLS gene has been demonstrated to be regulated by oncogene c-Myc, whereas GLS2 gene was identified as a target gene of p53 tumor suppressor. In glioblastomas (GBM, WHO grade IV), the most aggressive brain tumors, high levels of GLS and only traces or lack of GLS2 transcripts were found. Ectopic overexpression of GLS2 in human glioblastoma T98G cells decreased their proliferation and migration and sensitized them to the alkylating agents often used in the chemotherapy of gliomas. GLS silencing reduced proliferation of glioblastoma T98G cells and strengthen the antiproliferative effect evoked by previous GLS2 overexpression.

Glutamine as a mediator of ammonia neurotoxicity: A critical appraisal.

Ammonia is a major neurotoxin implicated in hepatic encephalopathy (HE). Here we discuss evidence that many aspects of ammonia toxicity in HE-affected brain are mediated by glutamine (Gln), synthesized in excess from ammonia and glutamate by glutamine synthetase (GS), an astrocytic enzyme. The degree to which Gln is increased in brains of patients with HE was found to positively correlate with the grade of HE. In animals with HE, a GS inhibitor, methionine sulfoximine (MSO), reversed a spectrum of manifestations of ammonia toxicity, including brain edema and increased intracranial pressure, even though MSO itself increased brain ammonia levels. MSO inhibited, while incubation with Gln reproduced the oxidative stress and cell swelling observed in ammonia-exposed cultured astrocytes. Recent studies have shown that astrocytes swell subsequent to Gln transport into mitochondria and its degradation back to ammonia, which then generates reactive oxygen species and the mitochondrial permeability transition. This sequence of events led to the formulation of the "Trojan Horse" hypothesis. Further verification of the role of Gln in the pathogenesis of HE will have to account for: (1) modification of the effects of Gln by interaction of astrocytes with other CNS cells; and (2) direct effects of Gln on these cells. Recent studies have demonstrated a "Trojan Horse"-like effect of Gln in microglia, as well as an interference by Gln with the activation of the NMDA/NO/cGMP pathway by ammonia as measured in whole brain, a process that likely also involves neurons.

Glutaminase: a hot spot for regulation of cancer cell metabolism?

Cancer cells re-program their metabolic machinery in order to satisfy their bioenergetic and biosynthetic requirements. A critical aspect of the re-programming of cancer cell metabolism involves changes in the glycolytic pathway (referred to as the "Warburg effect"). As an outcome of these changes, much of the pyruvate generated via the glycolytic pathway is converted to lactic acid, rather than being used to produce acetyl-CoA and ultimately, the citrate which enters the citric acid cycle. In order to compensate for these changes and to help maintain a functioning citric acid cycle, cancer cells often rely on elevated glutamine metabolism. Recently, we have found that this is achieved through a marked elevation of glutaminase activity in cancer cells. Here we further consider these findings and the possible mechanisms by which this important metabolic activity is regulated.

Molecular mechanism of inward rectifier potassium channel 2.3 regulation by tax-interacting protein-1.

Inwardly rectifying potassium channel 2.3 (Kir2.3) is specifically targeted on the basolateral membranes of epithelial and neuronal cells, and it thus plays an important role in maintaining potassium homeostasis. Tax-interacting protein-1 (TIP-1), an atypical PDZ-domain-containing protein, binds to Kir2.3 with a high affinity, causing the intracellular accumulation of Kir2.3 in cultured epithelial cells. However, the molecular basis of the TIP-1/Kir2.3 interaction is still poorly understood. Here, we present the crystal structure of TIP-1 in complex with the C-terminal Kir2.3-peptide (residues 436-445) to reveal the molecular details of the interaction between them. Moreover, isothermal titration calorimetry experiments show that the C-terminal Kir2.3-peptide binds much more strongly to TIP-1 than to mammalian Lin-7, indicating that TIP-1 can compete with mammalian Lin-7 to uncouple Kir2.3 from its basolateral membrane anchoring complex. We further show that the phosphorylation/dephosphorylation of Ser443 within the C-terminal Kir2.3 PDZ-binding motif RRESAI dynamically regulates the Kir2.3/TIP-1 association in heterologous HEK293T cells. These data suggest that TIP-1 may act as an important regulator for the endocytic pathway of Kir2.3.

Glutamine in neoplastic cells: focus on the expression and roles of glutaminases.

Glutamine is an important source of energy for neoplastic tissues, and products of its metabolism include, among others, glutamate (Glu) and glutathione (GSH), the two molecules that play a key role in tumor proliferation, invasiveness and resistance to therapy. Glutamine hydrolysis in normal and transforming mammalian tissues alike, is carried out by different isoforms of glutaminases, of which the two major are liver-type glutaminase (LGA) and kidney-type glutaminase (KGA). This brief review summarizes available data on the expression profiles and activities of these isoenzymes in different neoplastic tissues as compared to the tissues of origin, and dwells on recent work demonstrating effects of manipulation of glutaminase expression on tumor growth. A comment is devoted to the emerging evidence that LGA, apart from degrading Gln for metabolic purposes, is involved in gene transcription; its enforced overexpression in glioma cells was found to reduce their proliferation and migration.

New insights into brain glutaminases: beyond their role on glutamatergic transmission.

The synthesis of glutamate in brain must be exquisitely regulated because of its harmful potential giving rise to excitotoxic damage. In this sense, a stringent control based on multiple regulatory mechanisms should be expected to be exhibited by the biosynthetic enzymes responsible of glutamate generation, to assure that glutamate is only synthesized at the right place and at the right time. Glutaminase is considered as the main glutamate-producer enzyme in brain. Recently, novel glutaminase isoforms and extramitochondrial locations for these proteins have been discovered in the brain of mammals: identifying the function of each isozyme is essential for understanding the role of glutaminases in cerebral function. In addition, the interactome of glutaminases is starting to be uncovered adding a new level of regulatory complexity with important functional consequences, including selective and regulated targeting to concrete cellular locations. Finally, recent progress has identified glutaminase to be also present in astrocytes which precludes its classical consideration as a neuron-specific enzyme.

In vitro glutaminase regulation and mechanisms of glutamate generation in HIV-1-infected macrophage.

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis.

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase.

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.