Mechanistic Analysis of the Ubiquitin-Proteasome System

项目摘要/摘要 泛素-蛋白酶体系统(UPS)调节着数千种蛋白的活性、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几乎调节每一个细胞过程，包括细胞信号、细胞存活和细胞分裂。这个 UPS对生物学的广泛影响也对人类健康产生了重要影响，因为突变 UPS中的组件会导致癌症等疾病。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。出于这些原因， 对UPS的机械研究有可能揭示对生物监管的新见解，并可能 也有助于我们了解疾病的发病机制和治疗方法。 这一建议的一个主要重点是研究后期促进复合体/环体(APC)。这个 APC是一种多蛋白复合体，需要将泛素连接到调节细胞分裂的关键蛋白上。 在缺乏APC活性的情况下，细胞停滞在有丝分裂中。因为APC对细胞分裂是必不可少的，所以它可能 代表了治疗癌症的有用的药物靶点。出于这些原因，我们的实验室率先推出了 开发APC的小分子抑制剂，称为TAME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 更多关于APC的功能，通过识别在抑制剂存在下不能被降解的新蛋白质。 此外，我们计划确定影响细胞对APC抑制剂敏感性的基因。把这些放在一起 实验可能揭示APC的新功能，并可能有助于识别癌细胞中使 它们对APC抑制特别敏感。 APC是通过一些还不完全了解的复杂机制来管理的。 了解APC调节很重要，因为正确的APC激活时间对于 染色体分离。此外，我们不了解细胞如何控制其表达水平 不同的APC亚基。为了解决这些问题，我们计划重新构建依赖于APC的关键步骤 使用纯化的成分进行蛋白质分解。我们还计划定量地确定APC的组成 以及不同环境中的相关蛋白质，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。我们建议对这些蛋白质进行表征，以了解它们是如何降解的。我们特别是 对识别从这些底物中去除泛素的酶感兴趣，从而反对他们的 退化。这些研究将共同确定调节细胞内蛋白质稳定性的新途径，以及 可能为治疗疾病药物的开发提供新的起点。

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.