Active Glutaminase C Self-assembles into a Supratetrameric Oligomer That Can Be Disrupted by an Allosteric Inhibitor

• Published in: The Journal of biological chemistry Vol. 288; no. 39; pp. 28009 - 28020
• Main Authors: Ferreira, Amanda Petrina Scotá, Cassago, Alexandre, Gonçalves, Kaliandra de Almeida, Dias, Marília Meira, Adamoski, Douglas, Ascenção, Carolline Fernanda Rodrigues, Honorato, Rodrigo Vargas, de Oliveira, Juliana Ferreira, Ferreira, Igor Monteze, Fornezari, Camila, Bettini, Jefferson, Oliveira, Paulo Sérgio Lopes, Paes Leme, Adriana Franco, Portugal, Rodrigo Villares, Ambrosio, Andre Luis Berteli, Dias, Sandra Martha Gomes
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.09.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Algorithms; Allosteric Site; Cancer; Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Cross-Linking Reagents; Crystallography, X-Ray; Enzyme Inhibitors; Enzyme Inhibitors - chemistry; Enzyme Mechanisms; Gene Expression Regulation, Neoplastic; Glutaminase; Glutaminase - chemistry; Glutaminase - metabolism; Humans; Isoenzymes - chemistry; Metabolism; Microscopy, Electron, Transmission; Mutagenesis; Mutation; Phosphates - metabolism; Polymers - chemistry; Protein Conformation; Protein Multimerization; Protein Structure and Folding; Recombinant Proteins - metabolism; Warburg Effect; Enzyme Mechanisms; Enzyme Inhibitors; Warburg Effect; Metabolism; Glutaminase; Cancer
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M113.501346

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 321LRFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 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. Background: GAC supplies for increased metabolic needs of tumors because of exclusive localization and kinetic properties. Results: Higher than tetramer oligomers are the active form in in vitro and in cellular assays. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide disrupts oligomers. Conclusion: A novel molecular mechanism for GAC activation is proposed. Significance: The data affect the development of therapies targeting GAC in tumors, with emphasis on allosteric inhibitors.