Regulatory Enzymes and Systems in Cell Cycle Control

项目摘要/摘要 这项拟议中的研究探索了管理真核细胞分裂的有趣的调控系统。 周而复始。细胞周期控制系统基于主要的调节酶，包括依赖于细胞周期蛋白的 蛋白激酶(CDK)和一种称为后期促进复合体或环体的泛素连接酶(APC/C)。 随着细胞分裂的进行，顺序的CDK-Cyclin复合体修饰了数百个 蛋白质，导致S期染色体复制和染色体重复排列 有丝分裂纺锤体处于M期。然后，APC/C触发对几个关键调节器的破坏，释放 染色体分离和胞质分裂的戏剧性事件。一种关键的APC/C底物是Securin，一种 蛋白酶的抑制剂，分离酶；APC/C介导的Securin破坏释放分离酶，以切割 将复制的染色体连接在一起的粘附素复合体。细胞周期事件的精确计时需要 CDK和APC/C被激活并以特定顺序修改其目标。生物化学 这一顺序背后的机制是本申请中描述的研究的中心焦点。这项工作 我将探讨CDK和APC/C函数中一系列有趣的机制问题。萌芽中的研究 酵母将解决复杂的信息处理，这可以通过多位点磷酸化实现 CDK底物，重点是调控细胞周期蛋白基因表达的转录调控因子。多行 研究将探讨APC/C及其底物结合激活剂 子单位，控制对特定目标的破坏。一种新开发的单分子方法将提供 APC/C-底物相互作用的严格定量测量，提供了对 底物亲和力是细胞周期事件排序的基础。生化和结构方法 人类蛋白质将被用来解开APC/C激活亚基被加载到 APC/C由监护人TICRIC/CCT完成。将使用类似的方法来确定机制，方法是 哪些人类分离物识别其底物，以及Securin和其他调节机制 蛋白质抑制分离酶的催化功能。从这些研究中获得的信息将提供重要的 对控制细胞周期进程的新见解，从而将增强我们对疾病的理解， 例如癌症，细胞周期控制或染色体分离是有缺陷的。这些研究还将 阐明在细胞生物学和人类疾病中具有重要意义的一般调控机制。

PROJECT SUMMARY/ABSTRACT The proposed research explores the fascinating regulatory system that governs the eukaryotic cell division cycle. The cell-cycle control system is based on master regulatory enzymes that include the cyclin-dependent protein kinases (Cdks) and a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). As the cell progresses through the steps of cell division, sequential Cdk-cyclin complexes modify hundreds of proteins, leading to chromosome duplication in S phase and alignment of duplicated chromosome on the mitotic spindle in M phase. The APC/C then triggers destruction of several key regulators, unleashing the dramatic events of chromosome segregation and cytokinesis. One critical APC/C substrate is securin, an inhibitor of the protease, separase; APC/C-mediated securin destruction releases separase to cleave the cohesin complex holding duplicated chromosomes together. The precise timing of cell cycle events requires that Cdks and the APC/C are activated and modify their targets in a specific order. The biochemical mechanisms underlying this order are the central focus of the research described in this application. The work will explore a diverse array of interesting mechanistic problems in Cdk and APC/C function. Studies in budding yeast will address the complex information processing that can be achieved through multi-site phosphorylation of Cdk substrates, focusing on transcriptional regulators that govern cyclin gene expression. Multiple lines of investigation will address the mechanisms by which the APC/C, together with its substrate-binding activator subunit, controls the destruction of specific targets. A newly developed single-molecule approach will provide rigorous quantitative measurements of APC/C-substrate interactions, providing insight into the variations in substrate affinity that underlie the ordering of cell cycle events. Biochemical and structural approaches with human proteins will be used to unravel the mechanisms by which the APC/C activator subunit is loaded onto the APC/C by the chaperonin TRiC/CCT. Similar approaches will be used to determine the mechanisms by which human separase recognizes its substrates, and the mechanisms by which securin and other regulatory proteins inhibit separase catalytic function. The information gained from these studies will provide important new insights into the control of cell-cycle progression, and will thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control or chromosome segregation is defective. These studies will also illuminate general regulatory mechanisms of major importance throughout cell biology and human disease.