The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats.
ABSTRACT: On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.
Project description:Glutaminase, which converts glutamine to glutamate, is involved in Warburg effect in cancer cells. Two human glutaminase genes have been identified, GLS (GLS1) and GLS2. Two alternative transcripts arise from each glutaminase gene: first, the kidney isoform (KGA) and glutaminase C (GAC) for GLS; and, second, the liver isoform (LGA) and glutaminase B (GAB) for GLS2. While GLS1 is considered as a cancer therapeutic target, the potential role of GLS2 in cancer remains unclear. Here, we discovered a series of alkyl benzoquinones that preferentially inhibit glutaminase B isoform (GAB, GLS2) rather than the kidney isoform of glutaminase (KGA, GLS1). We identified amino acid residues in an allosteric binding pocket responsible for the selectivity. Treatment with the alkyl benzoquinones decreased intracellular glutaminase activity and glutamate levels. GLS2 inhibition by either alkyl benzoquinones or GLS2 siRNA reduced carcinoma cell proliferation and anchorage-independent colony formation, and induced autophagy via AMPK mediated mTORC1 inhibition. Our findings demonstrate amino acid sequences for selective inhibition of glutaminase isozymes and validate GLS2 as a potential anti-cancer target.
Project description: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.
Project description:Glutamine (Gln) metabolism, initiated by its degradation by glutaminases (GA), is elevated in neoplastic cells and tissues. In malignant glia-derived tumors, GA isoforms, KGA and GAC, coded by the GLS gene, are overexpressed, whereas the GLS2-coded GAB and LGA isoforms, are hardly detectable in there. Our previous study revealed that transfection of T98G glioblastoma cells with GAB reduced cell proliferation and migration, by a yet unknown mechanism not related to Gln degradation. The question arose how simultaneous overexpression of GAB and inhibition of KGA would affect glioblastoma cell growth. Here, we used siRNA to silence the expression of Gls in T98G cells which were or were not stably transfected with GAB (TGAB cells). In both T98G and TGAB cell lines, silencing of Gls with siRNAs targeted at different sequences decreased cell viability and proliferation in a different, sequence-dependent degree, and the observed decreases were in either cell line highly correlated with increase of intracellular Gln (r > 0.9), a parameter manifesting decreased Gln degradation. The results show that combination of negative modulation of GA isoforms arising from GLS gene with the introduction of the GLS2 gene product, GAB, may in the future provide a useful means to curb glioblastoma growth in situ. At the same time, the results underscore the critical role of Gln degradation mediated by KGA in the manifestations of aggressive glial tumor phenotype.
Project description:Alternative polyadenylation (APA) and alternative splicing (AS) provide mRNAs with the means to avoid microRNA repression through selective shortening or differential usage of 3'UTRs. The two glutaminase (GLS) mRNA isoforms, termed KGA and GAC, contain distinct 3'UTRs with the KGA isoform subject to repression by miR-23. We show that depletion of the APA regulator CFIm25 causes a strong shift to the usage of a proximal poly(A) site within the KGA 3'UTR and also alters splicing to favor exclusion of the GAC 3'UTR. Surprisingly, we observe that while miR-23 is capable of down-regulating the shortened KGA 3'UTR, it has only minor impact on the full-length KGA 3'UTR, demonstrating that additional potent negative regulation of GLS expression exists beyond this single microRNA targeting site. Finally, we show that the apoptosis induced upon down-regulation of the GAC isoform can be alleviated through concurrent reduction in CFIm25 expression, revealing the sensitivity of glutaminase expression to the levels of RNA processing factors. These results exemplify the complex interplay between RNA processing and microRNA repression in controlling glutamine metabolism in cancer cells.
Project description:Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability.
Project description:Kidney-type glutaminase (GLS) and liver-type glutaminase (GLS2) are dysregulated in many cancers, making them appealing targets for cancer therapy. However, their use as prognostic biomarkers is controversial and remains an active area of cancer research. Here, we performed a systematic multiomic analysis to determine whether glutaminases function as prognostic biomarkers in human cancers. Glutaminase expression and methylation status were assessed and their prominent functional protein partners and correlated genes were identified using various web-based bioinformatics tools. The cross-cancer relationship of glutaminases with mutations and copy number alterations was also investigated. Gene ontology (GO) and pathway analysis were performed to assess the integrated effect of glutaminases and their correlated genes on various cancers. Subsequently, the prognostic roles of GLS and GLS2 in human cancers were mined using univariate and multivariate survival analyses. GLS was frequently over-expressed in breast, esophagus, head-and-neck, and blood cancers, and was associated with a poor prognosis, whereas GLS2 overexpression implied poor overall survival in colon, blood, ovarian, and thymoma cancers. Both GLS and GLS2 play oncogenic and anti-oncogenic roles depending on the type of cancer. The varying prognostic characteristics of glutaminases suggest that GLS and GLS2 expression differentially modulate the clinical outcomes of cancers.
Project description:The gene GLS generates the phosphate activated glutaminase C (GAC) isoform by alternative splicing. GAC, compared to the other isoform, kidney-type glutaminase (KGA), has been characterized as more active and particularly important for cancer cell growth. Very little is known about post-translational modifications regulating GAC function. Hereby we describe the identification of a phosphorylation on the serine 95, located at the GLS N-terminus, a domain shared by both isoforms. A GAC phosphomimetic mutant (S95D) ectopically expressed in breast cancer cells presented decreased enzymatic activity, and its expression impacted on cell’s glutamine uptake, glutamate release and intracellular glutamate levels (compared to expressing wild type GAC) without changing GAC sub-cellular localization. Curiously, replacing S95 by an alanine in the ectopically expressed GAC (S95A) increased cell proliferation and migration. Taken together, these results reveal that GAC is post-translationally regulated by phosphorylation, which impacts on cancer phenotype.
Project description:Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.
Project description:Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.
Project description:Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.