Chemical Genetic and Biochemical Studies of Mitotic Proteolysis

描述（由申请人提供）：泛素-蛋白酶体系统调节包括细胞分裂在内的许多基本细胞过程。近年来抑制蛋白酶体的抗癌药物的发展表明，泛素-蛋白酶体系统可能提供许多新的治疗靶点。该项目的目标是开发新的泛素-蛋白酶体系统的化学抑制剂，并了解该途径如何调节细胞分裂。我们首先计划对后期促进复合体（APC）的新型小分子抑制剂进行表征，APC是一种泛素连接酶，对细胞分裂至关重要。APC抑制剂可能对治疗癌症有用，因为它们可能抑制细胞分裂或使细胞对抗有丝分裂药物（如紫杉醇）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负性调节轴突生长。我们对这些小分子的研究也将为APC如何被激活蛋白调节提供重要的新见解。我们研究的另一个目标是表征ubi抑素，这种化合物通过与泛素链结合来阻止泛素依赖的蛋白质水解。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，这将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的，也许更具有细胞渗透性的ubi抑素版本，增强这些化合物对该领域研究人员的效用，并加速泛素结合化合物作为候选药物的发展。我们研究计划的第三个目标是了解蛋白酶体如何识别泛素化底物进行降解。据推测，蛋白酶体降解需要一种特定类型的泛素链（通过泛素的赖氨酸-48连接）。我们对泛素化周期蛋白B1的研究表明，其他类型的泛素键可能足以靶向蛋白质降解。因此，我们计划通过纯化的蛋白酶体和复杂的细胞提取物来确定细胞周期蛋白B1降解所需的泛素化类型。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，我们想了解底物的定位如何影响泛素-蛋白酶体系统对其的降解。我们已经在细胞周期蛋白B1中发现了一个新的序列元件，该元件在有丝分裂期间将蛋白质招募到染色质上。我们计划鉴定将细胞周期蛋白B1招募到染色质上的蛋白，并确定细胞周期蛋白B1与染色质结合的功能后果。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在细胞周期中是如何被调节的。

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.