Function and regulation of the proteasome

描述（由申请人提供）：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着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基础，包括癌症、肌肉萎缩和神经病变。真核细胞中的大多数蛋白质降解是由26S蛋白酶体催化的，26S蛋白酶体是泛素系统中ATP依赖的蛋白酶。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。Specific Aim 1将描述26S蛋白酶体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。特异性Aim 2将利用生物化学定义的体外系统，确定ATP与AAA亚基结合介导26S蛋白酶体与蛋白酶（20S蛋白酶体）和调节性（PA700/19S）亚复合物的组装，以及组装时蛋白酶活性的激活的分子机制。这些实验将验证蛋白酶体组装和激活需要单独的ATP结合事件的假设，并确定单个AAA亚基在这些过程中的作用。这些实验将区分六个AAA子单元具有不同/专用角色或多个/冗余角色的可选模型。特异性Aim 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。这些实验将比较在结构稳定性和蛋白酶体靶向元件（如结构上定义的多泛素链）方面系统不同的模型底物变体的相对降解率。通过比较ATP水解和蛋白质水解的定性和定量要求，这些实验将解开ATP消耗在蛋白质水解亚过程中的多重作用，如底物结合、展开、易位和去泛素化。这些目标的完成将提供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。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着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基础，包括癌症、肌肉萎缩和神经病变。真核细胞中的大多数蛋白质降解是由26S蛋白酶体催化的，26S蛋白酶体是泛素系统中ATP依赖的蛋白酶。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。

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.