Chemical Genetic Studies of Mitosis

描述(由申请者提供)：我们研究项目的目标是 了解脊椎动物细胞中有丝分裂的退出是如何调节的，以及这是如何实现的 癌细胞的这一过程被打乱。真核细胞研究进展 周期由精心安排的蛋白质磷酸化和蛋白质控制。 退化。后期和有丝分裂退出依赖于泛素 安全蛋白和有丝分裂周期蛋白等调节因子的降解。这些蛋白质 以泛素化为靶标的多亚单位泛素连接酶称为 后期促进复合体或环体(APC)。我们的目标是研究APC是如何 通过寻找影响调节的化学物质在脊椎动物细胞中进行控制 或这个建筑群的活动。我们已经鉴定出几种化合物 稳定非洲爪哇卵提取液中的细胞周期蛋白B并通过 一种新的机制。我们的目标是确定这些基因的蛋白质靶点 使用生化表征和组合的抑制剂 基于亲和力的技术。我们还鉴定了一种化合物，它直接 在体外抑制APC的活性，我们将使用这种化合物来 研究APC在细胞内的调控。我们发现的有丝分裂抑制物之一 非洲爪哇细胞周期筛选矛盾地增加了细胞退出的速度 紫杉醇存在时的有丝分裂。我们将使用这种抑制剂来研究 细胞经历这种适应过程的机制。了解如何 退出有丝分裂和适应被调控将提供重要的见解 了解癌细胞对紫杉醇治疗的反应。

DESCRIPTION (provided by applicant): The goal of our research program is to understand how exit from mitosis is regulated in vertebrate cells and how this process is disrupted in cancer cells. Progression through the eukaryotic cell cycle is controlled by carefully timed protein phosphorylation and protein degradation. Anaphase and mitotic exit depend on the ubiquitin-dependent degradation of regulators such as securins and mitotic cyclins. These proteins are targeted for ubiquitination by a multisubunit ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC). Our goal is to study how APC is controlled in vertebrate cells by finding chemicals that affect the regulation or activity of this complex. We have identified several compounds that stabilize cyclin B in Xenopus egg extracts and block exit from mitosis through a novel mechanism. Our goal is to identify the protein targets of these inhibitors using a combination of biochemical characterization and affinity-based techniques. We have also identified a compound that directly inhibits the activity of the APC in vitro, and we will use this compound to study APC regulation in cells. One of the mitotic inhibitors discovered in our Xenopus cell cycle screen paradoxically increases the rate at which cells exit mitosis in the presence of taxol. We will use this inhibitor to study the mechanism by which cells undergo this adaptation process. Understanding how exit from mitosis and adaptation is regulated will provide important insights for understanding how cancer cells respond to taxol treatment.