RT Generic
T1 Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines
A1 Reverte López, María
AB CAD is a large multifunctional protein catalyzing the initial three steps in de novobiosynthesis of pyrimidine nucleotides in animals. Since pyrimidines are the buildingblocks of nucleic acids and the precursors of other key macromolecular substances, theup-regulation of CAD’s activity is essential for cell growth and proliferation, especially inneoplastic cells. Thus, CAD has been considered an attractive target for the developmentof anti-tumoral compounds. However, despite the central metabolic role and its therapeuticpotential, due to the lack of knowledge about its organization and the structureand function of its different enzymatic domains, no robust inhibitor that could be usedas anti-proliferative drug has been designed thus far. In this bachelor thesis, a researchstudy on CAD’s DHOase domain was conducted, particularly focused in understandingthe role of a flexible loop that appears to play a conserved catalytic role from E.coli tohumans. To examine the catalytic mechanism of this loop, a cloning approach was designedto generate a human DHOase construct bearing the equivalent flexible loop of theE.coli enzyme. This chimeric protein was expressed either in mammalian cells or in bacteriacultures and purified using different chromatographic techniques. Activity assays onboth the forward and reverse directions of the reaction were then performed in the chimerichuDHOase to estimate the turnover rate of the mutant. With a negligible enzymaticactivity (less than 2% of the wild type) the experiments here presented prove that, despitehaving a conserved functional role, the flexible loop of E.coli and human DHOases arenot interchangeable. Overall, the results confirmed the implication of the flexible loop inoligomerization and in the catalytic mechanism of DHOases, highlighting key differencesin the functioning of both mammalian and bacterial enzymes that will be exploited infuture work for the rational design of specific inhibitors against CAD.
YR 2018
FD 2018
LK https://hdl.handle.net/10016/29134
UL https://hdl.handle.net/10016/29134
LA eng
DS e-Archivo
RD 31 ago. 2024