Structural and Functional Analysis of Proteasome Core Particle Biogenesis

项目总结/摘要 蛋白质错误折叠是包括大多数神经退行性疾病在内的许多人类疾病的关键特征 和许多癌症。错误折叠蛋白的破坏主要由蛋白酶体介导， 多亚基复合物，这是迄今为止描述的最复杂的蛋白酶。蛋白酶体的活性位点 被隔离在一个桶形的圆柱形腔室中，称为核心颗粒（CP）。访问 蛋白酶体底物与CP的结合是由调节颗粒（RP）介导的，RP通过介导蛋白酶体底物与CP结合来识别蛋白酶体底物。 它们的泛素标签。RP展开，去泛素化，并将底物注入CP，在那里它被呈现 变成小肽。蛋白酶体的药理学抑制是一种已确立的抗癌疗法，大多数 尤其是多发性骨髓瘤。相反，增强蛋白酶体功能的可能性已经产生， 近年来，兴趣颇大。这种策略可能会改善由蛋白质错误折叠引起的疾病。 产生活性蛋白酶体的关键步骤是700 kDa 28亚基CP的组装， 在完整蛋白酶体组装之前，通过有序的多步途径发生， 五种专用的伴侣蛋白。CP成熟的结构分析受到以下挑战的阻碍： 分离和表征组装中间体，因为它们的丰度低和短暂性质。这里我们 假设确定的CP突变体可以富集装配中间体。我们已经开发出一种 这些突变体的亲和纯化和结构分析的生产性工作流程，并已 生成了八个高分辨率结构。在目标1中，我们将使用以下方法对CP突变体进行结构分析： 冷冻电子显微镜，加上详细的结构功能分析。在目标2中，我们将描述 长期以来已知但知之甚少的CP调节因子，称为PI 31/Fub 1。我们将尝试确定其 使用冷冻电子显微镜观察与CP复合的结构，并测试一些特定的假设 关于它的功能。在目标3中，我们将描述一种新的蛋白质， Rpn 4介导的蛋白酶体生物发生调节子的转录靶点，这似乎是一个新的 蛋白酶体相互作用蛋白。这一建议有望为蛋白酶体提供重要的见解 组装和整体功能，信息，这可能导致新的治疗策略的基础上，调节 蛋白酶体活性以治疗以蛋白质错误折叠为特征的疾病。

Project Summary/Abstract Protein misfolding is a key feature of many human diseases including most neurodegenerative diseases and many cancers. Destruction of misfolded proteins is largely mediated by the proteasome, a 2.5 MDa multisubunit complex which is the most sophisticated protease ever described. The proteasome's active sites are sequestered within a barrel-shaped cylindrical chamber, known as the core particle (CP). Access of substrates to the CP is mediated by the regulatory particle (RP), which recognizes proteasome substrates via their ubiquitin tags. The RP unfolds, deubiquitinates, and injects the substrate into the CP where it is rendered into small peptides. Pharmacologic inhibition of the proteasome is an established anti-cancer therapy, most notably in multiple myeloma. Conversely, the possibility of enhancing proteasome function has generated considerable interest in recent years. Such a strategy might ameliorate diseases caused by protein misfolding. A key step in the generation of active proteasomes is the assembly of the 700 kDa 28-subunit CP, which precedes assembly of the full proteasome and occurs by an ordered multistep pathway that requires the function of five dedicated chaperone proteins. Structural analysis of CP maturation has been hampered by challenges in isolating and characterizing assembly intermediates due to their low abundance and transitory nature. Here we hypothesized that defined CP mutants may be enriched for assembly intermediates. We have developed a productive work-flow for the affinity purification and structural analysis of these mutants, and have already generated eight high resolution structures. In Aim 1, we will carry out this structural analysis of CP mutants using Cryo-Electron Microscopy, coupled with detailed structure-function analyses. In Aim 2, we will characterize a long-known but poorly understood regulator of the CP known as PI31/Fub1. We will attempt to determine its structure in complex with the CP using Cryo-Electron Microscopy, and test a number of specific hypotheses regarding its function. In Aim 3, we will characterize a novel protein which is a previously unrecognized transcriptional target of the Rpn4-mediated proteasome biogenesis regulon, and which appears to be a new proteasome-interacting protein. This proposal is expected to provide significant insight into proteasome assembly and overall function, information which could lead to novel therapeutic strategies based on modulating proteasome activity to treat diseases characterized by protein misfolding.