Understanding the mechanism of activation and kinetic differences between the isoforms Kidney-type Glutaminase and Glutaminase C

Author(s)

Amanda Petrina Scotá Ferreira

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2013-12-13

Examining board members

Andre Luís Berteli Ambrosio; Artur Torres Cordeiro; Leandro Martínez

• Advisor: Andre Luís Berteli Ambrosio

Abstract

The energy production in cancer cells is abnormally dependent on aerobic glycolysis, resulting in increased consumption of glutamine to supply anabolic processes. The hydrolysis of glutamine is made by glutaminases, which have three isoforms identified in mammals: Kidney-type glutaminase (KGA), Glutaminase C (GAC) and the Liver-type Glutaminase (LGA). Although isoforms of KGA and GAC are identical in their catalytic domain, both have distinct catalytic efficiencies for glutamine. This project aims to find the reason for this difference catalytic, structurally studying these isoforms. The asymmetric unit of the crystallographic structure of GAC is composed by a tetramer, and this tetramerization is essential step for accessibility of the substrate glutamine, possibly stabilizing a "gating loop" in an open conformation. Site-directed mutations of residues from the "gating loop" (L321RFNKL326) disrupted the enzyme activity, which correlated directly with the ability of oligomerization: mutant which had lost activity were unable to form higher oligomers than tetramer. By transmission electron microscopy images using negative staining, we saw that the GAC forms filamentous structures in the presence of phosphate. Interestingly, a mutant in the region of the gating loop, called GAC.K325A, showed a very high catalytic efficiency and was able to form large oligomers. Furthermore, this mutant is insensitive to phosphate and BPTES. The mutant GAC.R322A lost activity and was unable to form polymers even in the presence of phosphate activator. Therefore, the oligomerization ability justifies the glutaminase activity exhibited by the protein: GAC - the higher glutaminase activity - formed the largest linear polymers, followed by KGA and, respectively, by LGA (low enzymatic efficiency and insensitive to inorganic phosphate). Furthermore, we showed the mechanism of BPTES inhibition is explained by disruption of oligomers. We present a mechanism for the formation of these oligomers, through a combination of deletions, point mutations, electron microscopy, molecular dynamics and docking. We simulated the acetylation of Lys311, and we found that this post-translational modification can maybe control enzymatic levels by down regulate the fiber formation. Using fluorescent labeling, we were able to define apparent dissociation constants of GAC and KGA isoforms. As the label interfered in the enzymatic activity, we believe that the technique produced artifacts unsuitable for the perception of differential interaction between isoform¿s superstructures. MDA-MB 231 cells with endogenous expression and knocked down GAC were used in cellular assays. Transfections with GAC.K325A-V5 proliferated more, consumed more glutamine from the culture medium and had higher turnover in glutaminase activity measurements. Collectively, these are evidence that the formation of superstructure glutaminases in vivo provides adaptive advantages to tumor cells and shows the importance of the enzyme as a therapeutic target (AU)

• FAPESP's process: 11/15107-0 - Understanding the kinetic differences between the Kidney-type Glutaminase and Glutaminase C isoforms
• Grantee: Amanda Petrina Scotá Ferreira
• Support Opportunities: Scholarships in Brazil - Master