Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/38382
Title: STRUCTURAL AND FUNCTIONAL BASIS OF GLUTAMINASE AS A NOVEL TARGET FOR ARRESTING CANCER CELLS METABOLISM
Authors: THANGAVELU K
Keywords: Warburg effect, RAS/MAPK, crystallography
Issue Date: 20-Nov-2012
Source: THANGAVELU K (2012-11-20). STRUCTURAL AND FUNCTIONAL BASIS OF GLUTAMINASE AS A NOVEL TARGET FOR ARRESTING CANCER CELLS METABOLISM. ScholarBank@NUS Repository.
Abstract: Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol- 2-yl) ethyl sulfide]. However, the structural and molecular basis of KGA inhibition and how the function of KGA is regulated in the cancer cells are still not clearly understood. We have studied the structure and function of KGA. Our studies revealed that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) andMek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2- K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA¿s active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling (Thangavelu et al., (2012) PNAS 109 (2), 7705-7710). In addition we studied the active site inhibition mechanism of KGA by determining the structure of KGA in complex with an active site inhibitor DON (6-Diazo-5-oxo-L-norlucine) and subsequent functional studies. Collectively our studies on allosteric and active site inhibition mechanism of KGA will help to design better drug lead compounds and strategies for the treatment of cancers addicted with glutamine metabolism.
URI: http://scholarbank.nus.edu.sg/handle/10635/38382
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