Functional Characterization: The human kinase Nek5 interferes negatively in cell death and the polyglutamylation process

Abstract

Protein kinases Nek (NIMA related kinases) are a highly conserved family of proteins involved in cell cycle progression. In humans have been identified Neks 11 (1-11), and for some of them the structure has been determined as well as the pathophysiological relevance. Several of Neks family proteins are products of genes related to diseases related to defects in cell cycle progression and the DNA repair mechanism during interphase, especially in different cancers. The Nek5 protein was identified, according to their genomic sequence in organisms such as Homo sapiens, Mus musculus, Bos taurus, among others. However, inherent characteristics of the protein remains poorly understood, as its function, structure and interactions. Therefore, the aim of this project is cloning, expression and purification Nek5 for biophysical testing and crystallization, aiming structural analysis. In parallel, will be conducted: 1) functional studies involving Nek5 silencing by RNA (iRNA) and overexpression in human cells; 2) identification of proteins that interact with Nek5 by mass spectrometry and yeast two-hybrid assay; and 3) thermal shift assays based on fluorescent and other in vitro assays to verify targets for Nek5 activity inhibition.