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1.
Proc Natl Acad Sci U S A ; 109(4): 1092-7, 2012 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-22228304

RESUMEN

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.


Asunto(s)
Glutaminasa/química , Glutaminasa/metabolismo , Mitocondrias/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Línea Celular Tumoral , Cristalización , Cristalografía por Rayos X , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Fosfatos/metabolismo , Unión Proteica , Dispersión del Ángulo Pequeño
2.
J Biol Chem ; 288(39): 28009-20, 2013 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-23935106

RESUMEN

The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop (321)LRFNKL(326) is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys(311) in humans, Lys(316) in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism.


Asunto(s)
Inhibidores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica , Glutaminasa/metabolismo , Multimerización de Proteína , Algoritmos , Sitio Alostérico , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular , Reactivos de Enlaces Cruzados , Cristalografía por Rayos X , Glutaminasa/química , Humanos , Isoenzimas/química , Microscopía Electrónica de Transmisión , Mutagénesis , Mutación , Fosfatos/metabolismo , Polímeros/química , Conformación Proteica , Proteínas Recombinantes/metabolismo
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