Publication: Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines
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2018
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2018-07-13
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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.
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Keywords
Biochemical analysis, Mutagenesis, ecombinant protein, Anti-tumoral target, Biochemistry