Abstract
A recurrent principle in cancer biology is the co-option of metabolic pathways to promote aberrant cell growth. In order to sustain an inherently proliferative phenotype, cancer cells are in constant process of adaptation to the types and levels of available nutrients in the tissue microenvironment they populate. It is now known that the ability of these cells to metabolically adapt is primarily associated with the activation of oncogenes or loss of tumor suppressors, which in turn very often result in the over expression of proteins isoforms that once were tightly regulated in normal cells. In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy, most cancer cells rely on aerobic glycolysis, even under normoxia, a phenomenon termed "Warburg effect". The amino acid glutamine and sugar glucose are two of the most important and versatile nutrients in this context. Both serve as the main sources of carbon skeletons (for nucleotide synthesis, amino acids and lipids), generation energy in the form of ATP and recycling of reducing agents, such as NADPH, all essential to the process of growth, biomass doubling and subsequent cell division. Renewed interest in cancer metabolism has prompted an innovative warfront against metabolic enzymes, aiming at the development of alternative and efficient therapeutic opportunities. Glutaminase, as an enzyme, is a key target in this sense and the need for new and accurate biochemical and structural information, in order to speed up and improve the development of successful therapies, is therefore essential. However, human glutaminases turn out to be more complex proteins, with a distinctive combination of additional motifs and functional domains, beyond its glutaminase domain, such as ankyrin repeats and nuclear receptor binding motifs. Hence, it would come as no surprise if the involvement of human glutaminases in processes taking place outside the mitochondrial boundaries is soon demonstrated. In this context, this project aims at the characterization of the interaction of glutaminases (Glutaminase C, kidney-type Glutaminase and liver-type Glutaminase) to completely novel partners recently identified in our lab through yeast two-hybrid assays. Initially, we plan to confirm the interaction in vitro by performing pull-down and gel filtration assays with the proteins expressed in recombinant system. We will then define the stoichiometry, the dissociation constants, and how theses interactions influence the glutaminase activity or the function of the interacting partner, whenever the assay for the protein is available. All the interactions that render stable complexes will be subject to crystallization trials and the obtained crystals will be diffracted and have their structure solved. (AU)
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