Function and Assembly of Eukaryotic Proteasome

描述（由申请人提供）：细胞中许多复杂的分子交易是由大型多亚基蛋白质机器催化的。通常，这些机器被组织成环形结构，形成一个中心通道或腔室，大分子通常通过完整的atp水解（atp酶）结构域或非共价相关的atp酶复合物进出这些腔室，这些atp酶复合物本身可能具有环形组织。26S蛋白酶体是真核细胞的中心胞内蛋白酶，是最复杂的环状ATP驱动机器之一。它由一个圆柱形的核心颗粒20S蛋白酶体组成，20S蛋白酶体包含一个中心的蛋白质水解室，而每个末端都有一个19S调节颗粒（RP）。RP包括六个不同的atp酶亚基，也可能在一个环状亚复合物中，它结合并展开蛋白质底物，并将它们驱动到20S蛋白酶体蛋白水解室。这种复杂的环状复合物如何在体内组装尚不清楚，对于具有四个异七聚体环的20S蛋白酶体的组装当然是如此。更不清楚的是~20亚基RP的组装机制。该应用程序的长期目标是描述体内蛋白酶体生物发生的途径。蛋白酶体已成为抗癌治疗和其他治疗的重要靶点。干扰其组装可为药物干预提供有益的新途径。本实验采用遗传、生化和生物物理方法相结合的方法，并以具有与人类复合物非常相似的26S蛋白酶体的模式真核生物酿酒酵母为中心。该项目的一个主要重点将是破译20S蛋白酶体在体内组装的途径，包括鉴定潜在的组装因子（Specific Aim 1）。20S蛋白酶体组装的步骤将在体外或细菌共表达系统中重建，并将测试假定的组装因子的机制（Specific Aim 2）。最后一组实验解决了RP如何组装的问题，这个问题目前只有最少的信息（具体目标3）。最近在PI实验室发现的潜在RP组装因子为这些研究提供了起点。

DESCRIPTION (provided by applicant): Many complex molecular transactions in the cell are catalyzed by large multisubunit protein machines. Often, these machines are organized into ring-shaped structures, creating a central channel or chamber, and macromolecules are moved into or out of these chambers, usually by integral ATP-hydrolyzing (ATPase) domains or by noncovalently associated ATPase complexes, which may themselves have a toroidal organization. The 26S proteasome, the central intracellular protease of eukaryotic cells, is among the most intricate of such ring-based ATP- driven machines. It consists of a cylindrical core particle, the 20S proteasome, which houses a central proteolytic chamber, and a 19S regulatory particle (RP) on each end. The RP includes six different ATPase subunits, also likely to be in a ring-shaped subcomplex, which binds and unfolds protein substrates and drives them into the 20S proteasome proteolytic chamber. How such complicated ring-shaped complexes are assembled in vivo is poorly understood, and this is certainly true for the assembly of the 20S proteasome, which has four heteroheptameric rings. Even less clear is the assembly mechanism of the ~20-subunit RP. The long-range goal of this application is to delineate the pathway(s) of proteasome biogenesis in vivo. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly could provide a useful new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, and biophysical methods and are centered on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major focus of the project will be on deciphering the pathway(s) by which the 20S proteasome assembles in vivo, including the identification of potential assembly factors (Specific Aim 1). Steps in 20S proteasome assembly will be reconstituted in vitro or in a bacterial co-expression system and the mechanisms of putative assembly factors will be tested (Specific Aim 2). A final set of experiments addresses the question of how the RP assembles, an issue for which there is only minimal information at present (Specific Aim 3). A potential RP assembly factor recently discovered in the PI's laboratory provides the starting point for these studies.