Molecular Control of Cell Proliferation

描述（由申请人提供）：本项目将探索真核细胞分裂周期各阶段的调控系统，重点是有丝分裂中染色体分离的控制。有丝分裂的最后事件是由一种鲜为人知的酶触发的，称为后期促进复合物或APC。APC是一种大的多亚基泛素连接酶，催化晚期有丝分裂事件的几个关键调节因子的泛素化。在拟议的研究中，生物化学和分子遗传学的方法将被用于芽殖酵母酿酒酵母（Saccharomyces cerevisiae）中，以剖析APC及其各种辅助成分识别其底物并组装多聚泛素链的酶学机制，该多聚泛素链将这些底物导向蛋白酶体进行破坏。提出了三个具体目标。第一个目标集中在APC如何识别其目标的重要问题上：诱变和详细的生化分析将被用来揭示三个相关APC亚基的功能，这些亚基包含保守的序列基序，tetratricopeptide repeat或TPR，它形成了一个参与底物募集的结合界面。第二个目标集中在与APC合作以帮助管理其功能的辅助酶：该实验室最近的研究表明，APC功能取决于两种泛素结合酶或E2，拟议的研究将探索这两种蛋白质的生化行为。进一步的工作将解决一个多聚泛素链延伸酶的功能，在初步研究中被确定为APC的潜在合作者。最后，在第三个目标中，将使用遗传和生物化学方法来了解APC靶标的泛素化状态如何受到细胞中去泛素化酶的调节。从这些研究中获得的知识将为控制染色体分离提供新的见解，染色体分离中的错误通常会导致发育问题和癌症进展。这些研究也可能阐明蛋白质泛素化的一般机制，这是一种在细胞生物学和人类疾病中具有重要意义的调节修饰。

DESCRIPTION (provided by applicant): This project will explore the regulatory system that governs progression through the stages of the eukaryotic cell division cycle, with an emphasis on the control of chromosome segregation in mitosis. The final events of mitosis are triggered by a poorly understood enzyme called the Anaphase-Promoting Complex or APC. The APC is a large, multisubunit ubiquitin ligase that catalyzes the ubiquitination of several key regulators of late mitotic events. In the proposed studies, biochemical and molecular genetic approaches will be used in the budding yeast Saccharomyces cerevisiae to dissect the enzymological mechanisms by which the APC, together with its various accessory components, recognizes its substrates and assembles polyubiquitin chains that direct those substrates to the proteasome for destruction. Three specific aims are proposed. The first aim focuses on the important question of how the APC recognizes its targets: mutagenesis and detailed biochemical analyses will be used to unveil the functions of three related APC subunits that contain a conserved sequence motif, the tetratricopeptide repeat or TPR, that forms a binding interface involved in substrate recruitment. The second aim centers on accessory enzymes that collaborate with the APC to help govern its function: recent studies from this laboratory have revealed that APC function depends on two ubiquitin-conjugating enzymes or E2s, and the proposed studies will explore the biochemical behavior of these two proteins. Additional work will address the function of a polyubiquitin chain-extending enzyme that was identified in preliminary studies as a potential collaborator of the APC. Finally, in the third aim, genetic and biochemical approaches will be used to understand how the ubiquitination state of APC targets is regulated by deubiquitinating enzymes in the cell. The knowledge gained from these studies will provide new insights into the control of chromosome segregation, errors in which often contribute to developmental problems and cancer progression. These studies are also likely to illuminate general mechanisms of protein ubiquitination, a regulatory modification of importance throughout cell biology and human disease.