Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines

Reverte López, María

Publication:
Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines

dc.contributor.advisor	Ramón Maiques, Santiago
dc.contributor.advisor	Guerrero Aspizua, Sara
dc.contributor.author	Reverte López, María
dc.contributor.departamento	UC3M. Departamento de Bioingeniería e Ingeniería Aeroespacial	es
dc.date.accessioned	2019-11-05T17:55:36Z
dc.date.available	2019-11-05T17:55:36Z
dc.date.issued	2018
dc.date.submitted	2018-07-13
dc.description.abstract	CAD is a large multifunctional protein catalyzing the initial three steps in de novo biosynthesis of pyrimidine nucleotides in animals. Since pyrimidines are the building blocks of nucleic acids and the precursors of other key macromolecular substances, the up-regulation of CAD’s activity is essential for cell growth and proliferation, especially in neoplastic cells. Thus, CAD has been considered an attractive target for the development of anti-tumoral compounds. However, despite the central metabolic role and its therapeutic potential, due to the lack of knowledge about its organization and the structure and function of its different enzymatic domains, no robust inhibitor that could be used as anti-proliferative drug has been designed thus far. In this bachelor thesis, a research study on CAD’s DHOase domain was conducted, particularly focused in understanding the role of a flexible loop that appears to play a conserved catalytic role from E.coli to humans. To examine the catalytic mechanism of this loop, a cloning approach was designed to generate a human DHOase construct bearing the equivalent flexible loop of the E.coli enzyme. This chimeric protein was expressed either in mammalian cells or in bacteria cultures and purified using different chromatographic techniques. Activity assays on both the forward and reverse directions of the reaction were then performed in the chimeric huDHOase to estimate the turnover rate of the mutant. With a negligible enzymatic activity (less than 2% of the wild type) the experiments here presented prove that, despite having a conserved functional role, the flexible loop of E.coli and human DHOases are not interchangeable. Overall, the results confirmed the implication of the flexible loop in oligomerization and in the catalytic mechanism of DHOases, highlighting key differences in the functioning of both mammalian and bacterial enzymes that will be exploited in future work for the rational design of specific inhibitors against CAD.	es
dc.description.degree	Ingeniería Biomédica (Plan 2010)	es
dc.identifier.uri	https://hdl.handle.net/10016/29134
dc.language.iso	eng	es
dc.rights	Atribución-NoComercial-SinDerivadas 3.0 España	*
dc.rights.accessRights	open access	es
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/3.0/es/	*
dc.subject.eciencia	Biología y Biomedicina	es
dc.subject.other	Biochemical analysis	es
dc.subject.other	Mutagenesis	es
dc.subject.other	ecombinant protein	es
dc.subject.other	Anti-tumoral target	es
dc.subject.other	Biochemistry	es
dc.title	Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines	es
dc.type	bachelor thesis	*
dspace.entity.type	Publication