Active glutaminase C self-assembles into a supratetrameric oligomer that can be disrupted by an allosteric inhibitor.
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.
From the Laboratórios Nacionais de Biociências e.
Amanda Petrina Scotá
AP
Ferreira
Alexandre
A
Cassago
Kaliandra de Almeida
Kde A
Gonçalves
Marília Meira
MM
Dias
Douglas
D
Adamoski
Carolline Fernanda Rodrigues
CF
Ascenção
Rodrigo Vargas
RV
Honorato
Juliana Ferreira
JF
de Oliveira
Igor Monteze
IM
Ferreira
Camila
C
Fornezari
Jefferson
J
Bettini
Paulo Sérgio Lopes
PS
Oliveira
Adriana Franco
AF
Paes Leme
Rodrigo Villares
RV
Portugal
Andre Luis Berteli
AL
Ambrosio
Sandra Martha Gomes
SM
Dias
D000465:Algorithms; D000495:Allosteric Site; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D005972:Glutaminase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D016296:Mutagenesis; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010710:Phosphates; D011108:Polymers; D011994:Recombinant Proteins; D005972:Glutaminase
Cancer; Enzyme Inhibitors; Enzyme Mechanisms; Glutaminase; Metabolism; Warburg Effect