Function and regulation of the proteasome

蛋白酶体的功能和调节

• 批准号: 8000778
• 负责人: George N. DeMartino
• 金额: $ 10.45万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-22 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000778
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; ATP-Dependent Proteases; Accounting; Achievement; Address; Affect; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Carboxypeptidase; Cell physiology; Chemicals; Collaborations; Complex; Consumption; Data; Disease; Doctor of Philosophy; Elements; Ensure; Escherichia coli; Eukaryotic Cell; Event; Foundations; Functional disorder; Goals; Hand; Health; Human Resources; Hydrolysis; In Vitro; Individual; Left; Louisiana; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Mutagenesis; Mutation; Peptide Hydrolases; Peptides; Play; Polyubiquitin; Preparation; Principal Investigator; Process; Proteasome Binding; Proteins; Proteolysis; Proteomics; Publications; Published Comment; Recombinant Proteins; Recombinants; Regulation; Relative (related person); Research; Research Personnel; Role; Saint Jude Children' s Research Hospital; Staging; Structure; Supervision; System; Testing; Training; Ubiquitin; Ubiquitination; Universities; Variant; base; crosslink; experience; improved; malignant muscle neoplasm; monomer; multicatalytic endopeptidase complex; mutant; neuropathology; programs; protein degradation; protein function; reconstitution; research study; response; success; wasting

DESCRIPTION (provided by applicant): The long term goal of this research is to comprehensively understand the mechanisms and regulation of intracellular protein degradation. Protein degradation regulates nearly every aspect of normal cellular function. Dysfunction of protein degradation underlies many diseases including cancer, muscle wasting, and neuropathologies. Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, the ATP dependent protease of the ubiquitin system. The immediate goal of this project is to determine the mechanisms by which ATP binding and hydrolysis mediate proteasome function. Specific Aim 1 will characterize biochemical features of ATP binding and hydrolysis by the 26S proteasome and define the relative qualitative and quantitative contributions of six different AAA ATPase subunits of the 26S proteasome to these processes. These experiments will provide essential new information about these subunits and support subsequent mechanistic studies on the roles of ATP and the AAA subunits in proteasome function. Specific Aim 2 will determine molecular mechanisms by which ATP binding to AAA subunits mediates both the assembly of the 26S proteasome from protease (20S proteasome) and regulatory (PA700/19S) sub complexes, and the activation of protease activity upon assembly, using biochemically defined in vitro systems. These experiments will test the hypothesis that proteasome assembly and activation require separate ATP binding events and define roles of individual AAA subunits in these processes. These experiments will distinguish between alternative models in which the six AAA subunits have either distinct/dedicated roles or multiple/redundant roles. Specific Aim 3 will define the role of ATP hydrolysis in 26S proteasome catalyzed protein degradation. These experiments will compare relative rates of degradation of variants of a model substrate that differ systematically in structural stability and proteasome targeting elements, such as structurally defined polyubiquitin chains. By comparing the qualitative and quantitative requirements for ATP hydrolysis and proteolysis, these experiments will deconvolute the multiple roles of ATP consumption in sub processes of proteolysis such as substrate binding, unfolding, translocation, and deubiquitylation. Completion of these aims will provide detailed information about mechanisms of proteolysis by the 26S proteasome and elucidate the fundamental role of ATP in mediating proteasome function. The long term goal of this research is to comprehensively understand the mechanisms and regulation of intracellular protein degradation. Protein degradation regulates nearly every aspect of normal cellular function. Dysfunction of protein degradation underlies many diseases including cancer, muscle wasting, and neuropathologies. Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, the ATP dependent protease of the ubiquitin system. The immediate goal of this project is to determine the mechanisms by which ATP binding and hydrolysis mediate proteasome function.

描述（申请人提供）：本研究的长期目标是全面了解细胞内蛋白质降解的机制和调控。蛋白质降解几乎调节正常细胞功能的各个方面。蛋白质降解功能障碍是许多疾病的根源，包括癌症、肌肉萎缩和神经病理学。真核细胞中的大多数蛋白质降解是由 26S 蛋白酶体（泛素系统的 ATP 依赖性蛋白酶）催化的。该项目的直接目标是确定 ATP 结合和水解介导蛋白酶体功能的机制。具体目标 1 将表征 26S 蛋白酶体的 ATP 结合和水解的生化特征，并定义 26S 蛋白酶体的六个不同 AAA ATP 酶亚基对这些过程的相对定性和定量贡献。这些实验将提供有关这些亚基的重要新信息，并支持后续关于 ATP 和 AAA 亚基在蛋白酶体功能中的作用的机制研究。具体目标 2 将使用生化定义确定 ATP 与 AAA 亚基结合介导 26S 蛋白酶体从蛋白酶（20S 蛋白酶体）和调节性 (PA700/19S) 亚复合物组装以及组装时激活蛋白酶活性的分子机制。体外系统。这些实验将检验蛋白酶体组装和激活需要单独的 ATP 结合事件的假设，并定义各个 AAA 亚基在这些过程中的作用。这些实验将区分六个 AAA 亚基具有不同/专用角色或多个/冗余角色的替代模型。具体目标 3 将定义 ATP 水解在 26S 蛋白酶体催化的蛋白质降解中的作用。这些实验将比较模型底物变体的相对降解率，这些变体在结构稳定性和蛋白酶体靶向元件（例如结构上确定的多聚泛素链）方面存在系统差异。通过比较 ATP 水解和蛋白水解的定性和定量要求，这些实验将解析 ATP 消耗在底物结合、解折叠、易位和去泛素化等蛋白水解子过程中的多重作用。完成这些目标将提供有关 26S 蛋白酶体蛋白水解机制的详细信息，并阐明 ATP 在介导蛋白酶体功能中的基本作用。这项研究的长期目标是全面了解细胞内蛋白质降解的机制和调控。蛋白质降解几乎调节正常细胞功能的各个方面。蛋白质降解功能障碍是许多疾病的根源，包括癌症、肌肉萎缩和神经病理学。真核细胞中的大多数蛋白质降解是由 26S 蛋白酶体（泛素系统的 ATP 依赖性蛋白酶）催化的。该项目的直接目标是确定 ATP 结合和水解介导蛋白酶体功能的机制。