Mechanistic Analysis of the Ubiquitin-Proteasome System

泛素-蛋白酶体系统的机理分析

• 批准号: 9898390
• 负责人: RANDALL W KING
• 金额: $ 41.83万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898390
• 关键词: Address; Biological; Biology; Cell Survival; Cell division; Cell physiology; Cells; Chromosome Segregation; Complex; Development; Disease; Enzymes; Genes; Goals; Health; Human; Learning; Malignant Neoplasms; Mitosis; Multiprotein Complexes; Mutation; Pathogenesis; Pathway interactions; Process; Proteins; Proteolysis; Regulation; Research; Signal Transduction; System; Ubiquitin; anaphase-promoting complex; cancer cell; cancer therapy; drug development; experimental study; inhibitor/antagonist; insight; interest; multicatalytic endopeptidase complex; new therapeutic target; novel; novel strategies; programs; protein protein interaction; reconstitution; small molecule inhibitor; targeted treatment; virtual

Project Summary / Abstract The ubiquitin-proteasome system (UPS) regulates the activity, localization and stability of thousands of proteins in the cell. By catalyzing the covalent attachment of ubiquitin to target proteins, the enzymes of the UPS regulate virtually every cellular process, including cell signaling, cell survival and cell division. The widespread influence of the UPS on biology also has important consequences for human health, as mutations in components of the UPS cause diseases such as cancer. Encouragingly, the vast number of enzymes and protein-protein interactions in the UPS are also providing new drug targets to treat disease. For these reasons, mechanistic studies of the UPS have the potential to reveal new insights into biological regulation and may also help us understand mechanisms of disease pathogenesis and therapy. A major focus of this proposal is to study the Anaphase-Promoting Complex/Cyclosome (APC). The APC is a multiprotein complex required for the attachment of ubiquitin to key proteins that regulate cell division. In the absence of APC activity, cells arrest in mitosis. Because the APC is essential for cell division, it may represent a useful drug target for the treatment of cancer. For these reasons, our lab has pioneered the development of small molecule inhibitors of the APC, called TAME and apcin. We have defined the mechanisms by which these inhibitors block APC activity. Here we propose to use these compounds to learn more about APC function, by identifying novel proteins that fail to be degraded in the presence of the inhibitors. In addition we plan to identify genes that influence the sensitivity of cells to APC inhibitors. Together these experiments may reveal new functions of the APC, and may help identify mutations in cancer cells that make them particularly sensitive to APC inhibition. The APC is regulated through a number of complex mechanisms that are not fully understood. Understanding APC regulation is important because proper timing of APC activation is essential for accurate chromosome segregation. Furthermore, we do not understand how the cell controls the level of expression of different APC subunits. To address these questions we plan to reconstitute key steps in APC-dependent proteolysis using purified components. We also plan to quantitatively determine the composition of the APC and associated proteins in different contexts to better understand its composition and function. Finally, we have developed a new approach to identify novel substrates of the ubiquitin-proteasome system. We propose to characterize these proteins to understand how they are degraded. We are particularly interested in identifying enzymes that remove ubiquitin from these substrates, thereby opposing their degradation. Together these studies will identify new pathways that regulate protein stability in the cell, and may provide new starting points for the development of drugs to treat disease.

项目总结/摘要 泛素-蛋白酶体系统（UPS）调节数千种蛋白酶的活性、定位和稳定性。 细胞中的蛋白质。通过催化泛素与靶蛋白的共价连接， UPS几乎调节每个细胞过程，包括细胞信号传导、细胞存活和细胞分裂。的 UPS对生物学的广泛影响也对人类健康产生重要影响， 不间断电源的组成部分会导致癌症等疾病。令人鼓舞的是，大量的酶和 UPS中的蛋白质-蛋白质相互作用也为治疗疾病提供了新的药物靶点。基于这些理由， UPS的机制研究有可能揭示生物调节的新见解， 也有助于我们了解疾病的发病机制和治疗。 该提案的一个主要重点是研究后期促进复合物/环体（APC）。的 APC是一种多蛋白复合物，用于将泛素连接到调节细胞分裂的关键蛋白。 在没有APC活性的情况下，细胞在有丝分裂中停滞。由于APC对细胞分裂至关重要， 代表了用于治疗癌症的有用的药物靶标。由于这些原因，我们的实验室开创了 开发APC的小分子抑制剂，称为驯服和apcin。我们已经定义了 这些抑制剂阻断APC活性的机制。在这里，我们建议使用这些化合物来学习 通过鉴定在抑制剂存在下不能降解的新蛋白质，更多地了解APC功能。 此外，我们计划确定影响细胞对APC抑制剂敏感性的基因。综合这些 实验可能揭示APC的新功能，并可能有助于识别癌细胞中的突变， 它们对APC抑制特别敏感。 APC通过许多复杂的机制进行调节，这些机制尚未完全了解。 理解APC调节是重要的，因为APC激活的适当时机对于准确的 染色体分离此外，我们不了解细胞如何控制表达水平， 不同的APC亚基。为了解决这些问题，我们计划重新构建依赖APC的 使用纯化的组分进行蛋白水解。我们还计划从数量上确定装甲运兵车的组成 以及不同背景下的相关蛋白质，以更好地了解其组成和功能。 最后，我们开发了一种新的方法来鉴定泛素-蛋白酶体的新底物 系统我们建议表征这些蛋白质，以了解它们是如何降解的。我们特别 对鉴定从这些底物中去除泛素的酶感兴趣，从而对抗它们的作用。 降解这些研究将共同确定调节细胞中蛋白质稳定性的新途径， 可能为开发治疗疾病的药物提供新的起点。