Chemical Genetic and Biochemical Studies of Mitotic Proteolysis

有丝分裂蛋白水解的化学遗传学和生化研究

• 批准号: 8061709
• 负责人: RANDALL W KING
• 金额: $ 35.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8061709
• 关键词: Affect; Antimitotic Agents; Antineoplastic Agents; Binding; Biochemical; Biochemical Reaction; Biological; CDC2 Protein Kinase; CDH1 gene; Cell Cycle; Cell Extracts; Cell division; Cell physiology; Cells; Chemicals; Chromatin; Complex; Conserved Sequence; Data; Development; Disease; Elements; Goals; Indium; Injury; Link; Lysine; Mediating; Mitosis; Mitotic; Nature; Paclitaxel; Pathway interactions; Permeability; Process; Protein Binding; Protein Kinase; Proteins; Proteolysis; Recruitment Activity; Regulation; Research; Research Personnel; Series; Signal Transduction; Structure; System; Tail; Testing; Tosylarginine Methyl Ester; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitination; Xenopus; anaphase-promoting complex; axon growth; base; cancer therapy; chemical genetics; chemical synthesis; cyclin B1; drug candidate; high throughput screening; improved; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; new therapeutic target; novel; preference; prevent; programs; protein degradation; receptor; reconstitution; research study; small molecule; tool; ubiquitin ligase; ubistatin A

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.

描述（由申请人提供）：泛素-蛋白酶体系统调节许多基本细胞过程，包括细胞分裂。近年来，蛋白酶体抑制剂的开发为泛素-蛋白酶体系统提供了新的治疗靶点。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。我们首先计划表征后期促进复合物（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抑制剂可用于治疗癌症，因为它们可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抑制剂可用于治疗涉及轴突损伤的疾病，因为APC负调节轴突生长。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泛素，通过结合泛素链来防止泛素依赖性蛋白水解的化合物。然而，泛素链结合的机制仍不清楚，我们计划合成新的泛素衍生物，这将使我们能够确定结合到泛素链的泛素的结构。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ubistatin版本，增强这些化合物对该领域研究人员的效用，并加速开发作为候选药物的泛素结合化合物。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降解的。已经假定，特定类型的泛素链（通过泛素的赖氨酸-48连接）是蛋白酶体降解所必需的。我们对泛素化细胞周期蛋白B1的研究表明，其他类型的泛素连接可能足以靶向蛋白质进行降解。因此，我们计划确定类型的泛素化所需的降解细胞周期蛋白B1的纯化蛋白酶体和复杂的细胞提取物。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它们通过泛素-蛋白酶体系统的降解。我们在细胞周期蛋白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募集到染色质中。我们计划确定招募细胞周期蛋白B1的染色质的蛋白质，并确定细胞周期蛋白B1与染色质协会的功能后果。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在细胞周期中的调节方式。