Biophysical and structural characterization of the interaction of kidney type glutaminase (KGA) and Peroxisome Proliferator-Activated Receptor gamma (PPARg)

Abstract

KGA (kidney-type glutaminase) is an isoform of mitochondrial glutaminase (GA) - enzyme responsible for catalyzing the conversion of glutamine to glutamate - encoded by the GLS1 gene. This reaction is the first step in the glutaminolysis pathway, a key process for tricarboxylic acid cycle (TCA) anapleurosis in order to provide biosynthetic precursors for the maintenance of the proliferative phenotype of tumor cells. PPARs (peroxisome proliferator-activated receptors) comprise a superfamily of nuclear receptors that control the expression of several genes involved in glucose and lipid metabolism. PPAR³ isoform is responsible for regulating the proliferation, differentiation and apoptosis of various human cancer cells. In addition to their important contributions to the tumor process widely described in the literature, KGA and PPAR³ seem to act as interaction partners, according to studies performed by our research group. Initially, yeast two-hybrid assays have indicated the N-terminal portion of the KGA and PPAR³-LBD (ligand binding domain) as probable interacting regions. Extensive in vitro assays are being performed in order to characterize the nature of this interaction in tumor cellular context. In parallel, the research project here proposed aims to characterize this interaction through structural and biophysical approaches. At first, we propose the identification of the minimal interacting regions of KGA and PPAR³, as well as the determination of the thermodynamic and energetic parameters of the interaction. Then, we propose the structural solution of KGA alone and in complex with PPAR³ using X-ray crystallography. Finally, we will perform in vivo analysis using FRET (Fluorescence Resonance Energy Transfer) microscopy. (AU)