Functional Analysis of the Proteasome Base

蛋白酶体碱基的功能分析

• 批准号: 7531901
• 负责人: Daniel J Finley
• 金额: $ 56.95万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7531901
• 关键词: ATP phosphohydrolase; Address; Affect; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological; Complex; Data; Deletion Mutation; Electron Microscopy; Eukaryota; Event; Family; Genetic; In Vitro; Individual; Ligand Binding; Ligands; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Methods; Mutation; N Domain; N-terminal; Nucleosome Core Particle; Pathway interactions; Pattern; Phenocopy; Play; Process; Property; Proteins; Proteomics; Recruitment Activity; Regulation; Role; Scaffolding Protein; Testing; Ubiquitin; Ubiquitination; base; multicatalytic endopeptidase complex; mutant; particle; protein degradation; scaffold

DESCRIPTION (provided by applicant): The proteasome, the most complex proteolytic assembly known, is a key mediator of biological regulation in eukaryotes. Ubiquitinated substrates are recognized by the 900-Kda regulatory particle (RP), then unfolded and translocated through a gated channel into the interior chamber of the core particle (CP) to be degraded. The key steps of recognition and unfolding are poorly understood, but appear to be critically dependent on a subassembly of the RP known as the base. The base is in direct contact with the CP, and is composed of 8 subunits, 6 of them ATPases of the AAA family. The present proposal aims to better understand how the base promotes protein degradation. Substrate recognition appears to be mediated by an ensemble of ubiquitin chain binding factors, including Rpnl0 and the UU proteins (such as Rad23 and Dsk2), all of which associate with the base. Still other factors, possibly intrinsic to the base, are also likely to be involved. In Aim 1 we will take a combined genetic, biochemical, and proteomic approach to resolve how these multiple pathways of substrate recognition are related: do they function redundantly, cooperatively, or independently? Are their functions overlapping for some substrates while being uniquely required to recognize others? The main focus of Aim 2 is an almost completely unstudied part of the base, the N-terminal domains of the 6 ATPases. Our hypothesis is that the N-domains play key roles in protein degradation, by projecting into the central compartment of the RP, where unfolding is thought to occur, and helping to promote this event by interacting directly with substrate. Among our proposed tests of this idea are attempts to screen genetically for mutations in the N-domains that differentially impair the degradation of one substrate but not another, at a step (presumably unfolding) that follows ubiquitin chain recognition. Aim 3 addresses our hypothesis that the largest subunit of the base, Rpnl, acts as a scaffold to recruit multiple factors to the proteasome, all of which are active on multiubiquitin chains. The proposed multiple functions of Rpnl will be dissected mainly by obtaining point mutants that specifically abrogate individual binding sites for postulated ligands such as Rad23, Rpnl0, and Ubp6. In summary, this proposal will combine biochemical, genetic, proteomic, and structural methods to address key questions concerning early steps in protein degradation by the proteasome.

描述（由申请人提供）：蛋白酶体是已知的最复杂的蛋白水解组件，是真核生物生物调节的关键介体。泛素化的底物通过900 kDa调节粒子（RP）识别，然后通过门控通道展开并转移到核心颗粒（CP）的内部室内（CP）。识别和展开的关键步骤知之甚少，但似乎严重取决于RP的子组件，称为碱基。基地与CP直接接触，由8个亚基组成，其中6个亚基是AAA家族的ATPases。本提案旨在更好地理解基础如何促进蛋白质降解。底物识别似乎是由泛素链结合因子的合奏介导的，包括RPNL0和UU蛋白（例如RAD23和DSK2），所有这些因子都与碱相关。还有其他因素，可能是基础的固有因素​​，也可能涉及。在AIM 1中，我们将采用一种组合的遗传，生化和蛋白质组学方法来解决这些底物识别的这些多个途径如何相关：它们是否有效，合作或独立地发挥作用？它们的功能是否重叠某些底物，同时被识别其他底物的独特需要？ AIM 2的主要重点是几乎完全未研究的部分，即6个ATPases的N末​​端域。我们的假设是，N域通过投射到RP的中心隔室中，在蛋白质降解中起关键作用，认为发生了发展，并通过直接与底物进行交互来帮助促进这一事件。在我们提出的关于这一想法的测试中，试图试图在遗传上筛选N域中的突变，这些突变会差异地损害了一种底物的降解，而不是另一种底物的降解，而不是另一种降解（大概是在泛素链链识别之后）。 AIM 3解决了我们的假设，即基数最大的亚基RPNL充当脚手架，将多种因素募集到蛋白酶体中，所有这些因素都在多泛素链上活跃。提出的RPNL的多个功能将主要通过获得特异性废除诸如RAD23，RPNL0和UBP6的个人结合位点的点突变体来解剖。总而言之，该提案将结合生化，遗传，蛋白质组学和结构方法，以解决有关蛋白酶体蛋白质降解早期步骤的关键问题。