Chemical Genetic and Biochemical Studies of Mitotic Proteolysis

描述(申请人提供)：泛素-蛋白酶体系统调节许多基本的细胞过程，包括细胞分裂。抑制蛋白酶体的抗癌药物的最新发展表明，泛素-蛋白酶体系统可能提供许多新的治疗靶点。这个项目的目标是开发泛素-蛋白酶体系统的新的化学抑制剂，并了解这一途径如何调节细胞分裂。我们首先计划表征后期促进复合体(APC)的新型小分子抑制剂，APC是一种对细胞分裂至关重要的泛素连接酶。APC抑制剂可能对癌症的治疗有用，因为它们可能抑制细胞分裂或使细胞对紫杉醇等抗分裂药物敏感。此外，APC抑制剂可能对涉及轴突损伤的疾病的治疗有用，因为APC负向调节轴突生长。我们对这些小分子的研究也将为激活蛋白如何调控APC提供重要的新见解。我们研究的另一个目标是表征泛素，一种通过与泛素链结合来阻止泛素依赖的蛋白分解的化合物。然而，泛素链结合的机制尚不清楚，我们计划合成新的泛素衍生物，使我们能够确定与泛素链结合的泛素的结构。这些结构信息将使我们能够合成更有效、也许更具细胞渗透性的泛素，增强这些化合物对该领域的研究人员的效用，并加速泛素结合化合物作为候选药物的开发。我们研究计划的第三个目标是了解泛素化底物是如何被蛋白酶体识别为降解的。有人认为，蛋白酶体的降解需要特定类型的泛素链(通过泛素的赖氨酸-48连接)。我们对泛素化的细胞周期蛋白B1的研究表明，其他类型的泛素连接可能足以靶向蛋白质的降解。因此，我们计划确定纯化的蛋白酶体和复杂细胞提取物降解细胞周期蛋白B1所需的泛素化类型。这些研究对于理解泛素化和泛素链识别的小分子抑制剂如何干扰降解至关重要。最后，我们想要了解底物的定位如何影响泛素-蛋白酶体系统对它们的降解。我们已经在细胞周期蛋白B1中发现了一个新的序列元件，它在有丝分裂过程中将蛋白质招募到染色质中。我们计划鉴定将细胞周期蛋白B1招募到染色质的蛋白质，并确定细胞周期蛋白B1与染色质相关的功能后果。总之，这些研究将为研究泛素-蛋白酶体途径提供新的化学工具，并将帮助我们了解该途径的关键底物--细胞周期蛋白B1是如何在细胞周期中被调节的。

DESCRIPTION (provided by applicant): The ubiquitin-proteasome system regulates many essential cellular processes including cell division. The recent development of cancer drugs that inhibit the proteasome suggests that the ubiquitin-proteasome system may provide many new therapeutic targets. The goal of this project is to develop new chemical inhibitors of the ubiquitin-proteasome system, and to understand how this pathway regulates cell division. We first plan to characterize novel small molecule inhibitors of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that is essential for cell division. APC inhibitors may be useful for the treatment of cancer, because they may inhibit cell division or sensitize cells to antimitotic drugs such as taxol. Additionally, APC inhibitors may be useful for the treatment of diseases that involve axonal injury, because the APC negatively regulates axon growth. Our studies of these small molecules will also provide important new insights into how the APC is regulated by activator proteins. Another goal of our studies is to characterize ubistatins, compounds that prevent ubiquitin- dependent proteolysis by binding to ubiquitin chains. However, the mechanism of ubiquitin chain binding remains unclear, and we plan to synthesize new ubistatin derivatives that will allow us to determine the structure of ubistatins bound to ubiquitin chains. The structural information will enable us to synthesize more potent and perhaps more cell-permeable versions of ubistatins, enhancing the utility of these compounds to investigators in the field, and accelerating the development of ubiquitin-binding compounds as drug candidates. A third goal of our research program is to understand how ubiquitinated substrates are recognized for degradation by the proteasome. It has been assumed that a specific type of ubiquitin chain (linked through lysine-48 of ubiquitin) is required for degradation by the proteasome. Our studies of ubiquitinated cyclin B1 suggest that other types of ubiquitin linkages may be sufficient to target the protein for degradation. We therefore plan to determine the types of ubiquitination that are required for degradation of cyclin B1 by purified proteasomes and in complex cell extracts. These studies are critical for understanding how small molecule inhibitors of ubiquitination and ubiquitin chain recognition may perturb degradation. Finally, we want to understand how the localization of substrates affects their degradation by the ubiquitin-proteasome system. We have identified a novel sequence element in cyclin B1 that recruits the protein to chromatin during mitosis. We plan to identify the protein that recruits cyclin B1 to chromatin, and to determine the functional consequences of cyclin B1 association with chromatin. Together these studies will provide new chemical tools for studying the ubiquitin-proteasome pathway, and will help us understand how a critical substrate of this pathway, cyclin B1, is regulated during the cell cycle.