Functional Analysis of the Proteasome Base

蛋白酶体碱基的功能分析

• 批准号: 8080023
• 负责人: Daniel J Finley
• 金额: $ 27.03万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080023
• 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(申请人提供)：蛋白酶体是已知的最复杂的蛋白分解组合，是真核生物生物调节的关键媒介。泛素化的底物被900-KDA调节颗粒(RP)识别，然后展开并通过门控通道转移到核心颗粒(CP)的内室进行降解。识别和展开的关键步骤人们知之甚少，但似乎严重依赖于被称为碱基的RP的一个子集。该碱基与CP直接接触，由8个亚基组成，其中6个是AAA家族的ATPase。本提案旨在更好地了解碱基是如何促进蛋白质降解的。底物识别似乎是由一系列泛素链结合因子介导的，包括Rpnl0和Uu蛋白(如RAD23和Dsk2)，所有这些都与碱基相关。还有其他因素，可能是基础所固有的，也可能涉及其中。在目标1中，我们将采用遗传学、生物化学和蛋白质组学相结合的方法来解决这些底物识别的多个途径是如何相关的：它们是冗余地、协同地还是独立地发挥作用？它们的功能是否与某些底物重叠，而识别另一些底物是唯一需要的？目标2的主要焦点是碱基的一个几乎完全未被研究的部分，即6个ATPase的N-末端结构域。我们的假设是，N-结构域在蛋白质降解中发挥关键作用，通过投射到RP的中央隔室，在那里被认为是发生展开的地方，并通过直接与底物相互作用帮助促进这一事件。在我们提出的对这一想法的测试中，有一项是试图从基因上筛选N-结构域中的突变，这些突变对一种底物的降解有不同的损害，但对另一种底物的降解没有影响，这一步骤(假设是展开)是在泛素链识别之后进行的。目的3解决了我们的假设，即碱基最大的亚基Rpn1作为支架向蛋白酶体招募多种因子，所有这些因子都活跃在多泛素链上。Rpn1的多重功能将主要通过获得点突变来进行剖析，这些点突变可以特异性地消除假设的配体如RAD23、Rpn10和Ubp6的单个结合位点。综上所述，这项提案将结合生化、遗传学、蛋白质组学和结构方法来解决与蛋白质酶体降解蛋白质的早期步骤有关的关键问题。