Function and Assembly of Eukaryotic Proteasomes

真核蛋白酶体的功能和组装

• 批准号: 8811972
• 负责人: Mark W Hochstrasser
• 金额: $ 32.42万
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8811972
• 关键词: 26S proteasome; ATP phosphohydrolase; Address; Binding; Biochemical; Biogenesis; Biological; Biological Process; Cell Nucleus; Cells; Complex; DNA biosynthesis; Diabetes Mellitus; Disease; Escherichia coli; Eukaryota; Fluorescence Microscopy; Genetic; Genomics; Goals; Grant; Growth; Health; Housing; Human; Imagery; In Vitro; Intervention; Length; Malignant Neoplasms; Methodology; Methods; Modeling; Molecular Chaperones; Movement; Multienzyme Complexes; Neurodegenerative Disorders; Nucleosome Core Particle; Oxidative Stress; Pathway interactions; Pharmacologic Substance; Physiological; Process; Proline; Proteins; RNA Processing; Regulation; Role; Saccharomyces cerevisiae; Shapes; Specificity; Structure; Testing; Tissues; Tumor Suppressor Proteins; Variant; Yeasts; base; biophysical techniques; cancer therapy; human disease; in vivo; multicatalytic endopeptidase complex; mutant; novel; novel strategies; particle; protein degradation; reconstitution; research study; response

DESCRIPTION (provided by applicant): Multiprotein machines control many complex biological processes, such as DNA replication, RNA processing, and protein degradation. The 26S proteasome is responsible for the majority of protein degradation in eukaryotes and is essential for numerous cellular activities. Aberrant proteasomal activity impacts many human diseases including cancer, neurodegenerative disorders, and diabetes. Like many multisubunit catalytic complexes, the proteasome forms ring-shaped structures through which a substrate is passed, allowing multiple enzymatic activities to act sequentially on the substrate. The proteasome consists of a cylindrical 20S proteasome core particle (CP) with a 19S regulatory particle (RP) on one or both ends. Each RP can be divided into lid and base subcomplexes. The CP has two outer heteroheptameric 1 rings that sandwich a pair of 2 rings, which house the central proteolytic chamber. The RP base includes six different ATPases that form a hexameric ring; the RP binds and unfolds substrates and drives them into the CP interior. How these large complexes are assembled in vivo is poorly understood. This is true for the CP and even more so for the ~19-subunit RP. Proteasome assembly appears to be an ordered process conserved across species. As proteasomes are highly abundant, assembly must occur with high fidelity to avoid excessive accumulation of nonproductive and potentially toxic off-pathway intermediates. Both CP and RP assembly depend on dedicated assembly chaperones. The long-range goal of this grant is to delineate the pathways of eukaryotic 26S proteasome biogenesis. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly will provide a new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, cell biological and biophysical methods and are focused primarily on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major goal of this first renewal will be on deciphering the assembly-promoting mechanisms of a CP assembly factor (Aim 1) and four RP assembly chaperones (RACs) (Aim 2). Both sets of factors were discovered during the past cycle of this grant. Aim 3 is directed toward determining what intermediates of the RP lid form in vivo and whether these serve as assembly intermediates. Lid-base joining will be a key step to be analyzed. Finally, Aim 4 considers the question of where in the cell the proteasome assembles and how proteasomes relocalize within the cell in response to changes in growth conditions.

描述（申请人提供）：多蛋白机器控制许多复杂的生物过程，例如DNA复制、RNA加工和蛋白质降解。26 S蛋白酶体负责真核生物中的大多数蛋白质降解，并且对于许多细胞活动是必需的。异常的蛋白酶体活性影响许多人类疾病，包括癌症、神经退行性疾病和糖尿病。像许多多亚基催化复合物一样，蛋白酶体形成环状结构，底物通过该环状结构，允许多种酶活性依次作用于底物。蛋白酶体由一个圆柱形的20 S蛋白酶体核心颗粒（CP）和一端或两端的19 S调节颗粒（RP）组成。每个RP可以分为盖和基础亚复合物。CP具有两个外杂七聚体1环，其夹着一对2环，其容纳中心蛋白水解室。RP碱基包括六种不同的ATP酶，它们形成一个六聚体环; RP结合并展开底物，并将其驱动到CP内部。这些大的复合物如何在体内组装还知之甚少。这对于CP是真实的，对于~19-亚基RP更是如此。蛋白酶体组装似乎是一个有序的过程中保守的物种。由于蛋白酶体是高度丰富的，组装必须以高保真度发生，以避免非生产性和潜在毒性的途径外中间体的过度积累。CP和RP组装都依赖于专用的组装分子伴侣。这项资助的长期目标是描绘真核生物26 S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。干扰其组装将为药物干预提供新的途径。拟议的实验使用遗传，生物化学，细胞生物学和生物物理学方法的组合，并主要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。第一次更新的主要目标是破译CP组装因子（Aim 1）和四个RP组装分子伴侣（RAC）（Aim 2）的组装促进机制。这两组因素都是在本赠款的上一个周期中发现的。目的3是针对确定RP盖在体内形成的中间体以及这些中间体是否用作组装中间体。盖基连接将是要分析的关键步骤。最后，目标4考虑了蛋白酶体在细胞中哪里组装以及蛋白酶体如何在细胞内重新定位以响应生长条件的变化的问题。