Biochemical characterization of cell cycle deubiquitinating enzymes

细胞增殖由一个复杂的生化反应网络来调节，以确保每个细胞分裂步骤都以正确和正确的顺序进行。在这些反应中，有一种被称为泛素-蛋白酶体系统(UPS)的酶系统对细胞调节蛋白的定时降解。在这个系统中，名为E3泛素连接酶的酶复合体将多个拷贝的泛素添加到底物蛋白上，标记它以供蛋白酶体快速降解。另一类酶被鉴定为去泛素酶(DUBS)，它从底物蛋白中去除泛素，与E3连接酶的作用相反。E3连接酶和DUBS活性的平衡决定了底物蛋白的稳定性，在细胞分裂周期的调控中起着关键作用。这种平衡的放松会导致不受控制的细胞增殖和遗传不稳定。*目前缺乏参与细胞分裂调控的E3连接酶和DUBS网络的全面图景。利用大规模的功能基因组学方法，我们的实验室最近发现了控制关键细胞周期调节蛋白的稳定性和表达水平的新的DUB。这项研究计划的目的是阐明这些酶作用的分子机制，确定它们的细胞靶标，并确定它们对细胞增殖的影响。我们将使用互补的生物化学、遗传、定量蛋白质组学和细胞生物学方法来解决这些问题。总的来说，这些研究将有助于更好地理解蛋白质降解和UPS在控制细胞分裂及其在人类疾病中的故障中的作用。**

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.**