Biochemical characterization of cell cycle deubiquitinating enzymes

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Cell proliferation is regulated by a complex network of biochemical reactions that ensure that each cell division step is performed correctly and in proper sequence. Among these reactions is the timed degradation of cellular regulatory proteins by an enzymatic system known as the ubiquitin-proteasome system (UPS). In this system, enzyme complexes named E3 ubiquitin ligases add multiple copies of ubiquitin to a substrate protein, tagging it for rapid degradation by the proteasome. Another family of enzymes identified as deubiquitinating enzymes (DUBs) remove ubiquitin from substrate proteins, opposing the action of E3 ligases. The balance in the activities of E3 ligases and DUBs determine the stability of substrate proteins and plays a key role in the regulation of the cell division cycle. Deregulation of this balance leads to uncontrolled cell proliferation and genetic instability. A comprehensive picture of the network of E3 ligases and DUBs involved in the regulation of cell division is currently lacking. Using large-scale functional genomic approaches, our laboratory has recently identified novel DUBs that control the stability and expression level of key cell cycle regulatory proteins. The objectives of this research proposal are to elucidate the molecular mechanisms underlying the effect of these enzymes, to identify the repertoire of their cellular targets, and to define their impact on cell proliferation. We will use a combination of complementary biochemistry, genetic, quantitative proteomics and cell biology approaches to address these questions. Collectively, these studies will contribute to a better understanding of the role of protein degradation and the UPS in the control of cell division and its malfunction in human diseases.