Function and Assembly of Eukaryotic Proteasome

真核蛋白酶体的功能和组装

• 批准号: 7759509
• 负责人: Mark W Hochstrasser
• 金额: $ 28.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7759509
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Address; Affect; Binding; Biochemical; Biogenesis; Cells; Complex; Escherichia coli; Eukaryota; Eukaryotic Cell; Genetic; Goals; Housing; Human; In Vitro; Intervention; Laboratories; Methods; Modeling; Molecular; Molecular Machines; Names; Nucleosome Core Particle; Pathway interactions; Peptide Hydrolases; Pharmacologic Substance; Principal Investigator; Proteins; Recombinant Proteins; Research; Role; Saccharomyces cerevisiae; Shapes; Staging; Structure; System; Testing; Yeasts; base; cancer therapy; in vivo; macromolecule; multicatalytic endopeptidase complex; mutant; novel strategies; particle; programs; reconstitution; research study

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.

描述（由申请人提供）：细胞中许多复杂的分子交易由大型多亚基蛋白质机器催化。通常，这些机器被组织成环形结构，产生中央通道或腔室，并且大分子通常通过整合的ATP水解（ATP酶）结构域或通过非共价缔合的ATP酶复合物移入或移出这些腔室，所述ATP酶复合物本身可能具有环形组织。26 S蛋白酶体，真核细胞的中心细胞内蛋白酶，是这种基于环的ATP驱动的机器中最复杂的。它由一个圆柱形的核心颗粒，20 S蛋白酶体，其中包含一个中央蛋白水解室，和一个19 S调节颗粒（RP）的每一端。RP包括六个不同的ATP酶亚基，也可能是一个环形的亚复合物，结合和展开蛋白质底物，并将它们驱动到20 S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还知之甚少，这对于具有四个杂七聚体环的20 S蛋白酶体的组装来说无疑是正确的。更不清楚的是~20-亚基RP的组装机制。本申请的长期目标是描绘体内蛋白酶体生物合成的途径。蛋白酶体已成为抗癌治疗和其他治疗的重要靶点。干扰其组装可能为药物干预提供一个有用的新途径。拟议的实验使用遗传，生物化学和生物物理方法的组合，并集中在模型真核生物酿酒酵母，它有一个26 S蛋白酶体非常相似的人类复杂。该项目的一个主要重点将是破译20 S蛋白酶体在体内组装的途径，包括识别潜在的组装因子（具体目标1）。将在体外或细菌共表达系统中重建20 S蛋白酶体组装步骤，并检测推定组装因子的机制（具体目标2）。最后一组实验解决了RP如何组装的问题，目前只有最少的信息（具体目标3）。PI实验室最近发现的一个潜在的RP组装因子为这些研究提供了起点。