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Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

ABSTRACT: 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.

SUBMITTER: Cassago A

PROVIDER: S-EPMC3268272 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

Cassago Alexandre A Ferreira Amanda P S AP Ferreira Igor M IM Fornezari Camila C Gomes Emerson R M ER Greene Kai Su KS Pereira Humberto M HM Garratt Richard C RC Dias Sandra M G SM Ambrosio Andre L B AL

Proceedings of the National Academy of Sciences of the United States of America 20120106 4

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 ...[more]

PMID: 22228304

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Project description:Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCβII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCβII specific inhibitor peptide βIIV5-3-treated IR. Vehicle or βIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCβII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 μM) to evaluate the inhibition of GLS1 on neuronal viability. PKCβII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by βIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCβII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.

Dataset Information

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

Publications

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

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