Regulation of Proteasome Activity

蛋白酶体活性的调节

• 批准号: 10406057
• 负责人: Daniel J Finley
• 金额: $ 49.72万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10406057
• 关键词: Affect; Animal Model; Binding; Caenorhabditis elegans; Cells; Complex; Deubiquitinating Enzyme; Disease; Enzymatic Biochemistry; Enzymes; Eukaryota; Genetic; Impairment; Laboratories; Longevity; Mediating; Methods; Nerve Degeneration; Output; Pathway interactions; Peptide Hydrolases; Perception; Proteins; Proteomics; Regulation; Reporting; Role; Specificity; Stress; Structure; Substrate Specificity; System; Time; Ubiquitin; Work; Yeasts; fascinate; genetic analysis; interest; multicatalytic endopeptidase complex; mutant; polyglutamine; proteostasis; receptor; reconstitution; recruit; single molecule; structural biology; ubiquitin ligase

PROJECT SUMMARY / ABSTRACT The focus of my laboratory’s efforts is the proteasome, the protease that degrades ubiquitin-protein conjugates. That the proteasome is the central activity within the ubiquitin-proteasome system (UPS) was known from the time the UPS was charted, but its significance was not fully recognized at the time because the proteasome was presumed, incorrectly, to be unregulated and passive–essentially a dumb enzyme. But this perception has been overturned by the progressive identification of diverse mechanisms that finely regulate proteasome synthesis, turnover, localization, substrate specificity, and specific activity. These mechanisms are of special interest because they provide the means for global control of UPS output. In parallel, evidence of the importance of proteasome activity in disease has accumulated. For example, even a modest elevation of proteasome levels substantially increases the lifespan of D. melanogaster and C. elegans, as well as their ability to withstand stresses such as the expression of toxic polyQ proteins. A fascinating mode of proteasome regulation is that involving the dynamic reconfiguration of ubiquitin chains on a substrate at the proteasome. Two such chain-editing factors are highly active as well as conserved across eukaryotes: Ubp6/USP14 and Hul5/UBE3C. Ubp6 is a deubiquitinating enzyme and Hul5 is a ubiquitin ligase, and they work in opposition to one another; Ubp6 will remove ubiquitin groups added to the substrate by Hul5. Both are recruited to proteasomes when the UPS is impaired or challenged. The specificity of Ubp6 is remarkable in that it acts only on substrates that carry multiple ubiquitin chains. We will characterize this specificity further and investigate its mechanistic basis. We have reported that Hul5 functions as an E4 on the proteasome–it ubiquitinates proteins that are already ubiquitinated. We will focus now on how this E4 activity promotes the processivity of the proteasome, as Hul5 appears to be the main regulator of processivity. We will reconstitute the processivity effect in a purified system and use highly specific mutants in Hul5 in single-molecule analysis. We will also investigate the mechanisms by which Ubp6 and Hul5 are controlled by stress. The third major factor recruited to proteasomes under proteostasis stress is Ecm29, which has the unique feature of binding both the RP and CP. Ecm29 regulates both the activity and assembly of the proteasome, most likely by bridging these complexes. Our studies will focus on how Ecm29 is recruited to faulty proteasomes and how it affects their structure and stability. Finally, substrate recognition by the proteasome is mediated by six distinct ubiquitin receptors. However, our detailed genetic analysis in yeast indicates the existence of at least one additional, unknown ubiquitin receptor within the proteasome. We will attempt to identify this receptor, and once we generate suitably precise mutants we will explore this receptor’s role in substrate recognition and processing. In summary, we propose, by applying the methods of genetics, enzymology, structural biology, and quantitative global proteomics, to elucidate major pathways of proteasome function and regulation.

项目概要/摘要 我实验室的工作重点是蛋白酶体，即降解泛素蛋白的蛋白酶 共轭物。众所周知，蛋白酶体是泛素蛋白酶体系统 (UPS) 中的核心活性 从 UPS 制定之日起，其重要性在当时并未得到充分认识，因为 人们错误地认为蛋白酶体是不受调控的、被动的——本质上是一种愚蠢的酶。但这 各种精细调节机制的逐步识别已经颠覆了人们的看法 蛋白酶体合成、周转、定位、底物特异性和比活性。这些机制是 之所以特别令人感兴趣，是因为它们提供了对 UPS 输出进行全局控制的方法。与此同时，证据表明 蛋白酶体活性在疾病中的重要性不断增加。例如，即使是适度的提升 蛋白酶体水平显着延长黑腹果蝇和线虫的寿命及其能力 承受压力，例如有毒的polyQ蛋白的表达。蛋白酶体的迷人模式 调节涉及蛋白酶体底物上泛素链的动态重构。二 此类链编辑因子在真核生物中高度活跃且保守：Ubp6/USP14 和 Hul5/UBE3C。 Ubp6 是一种去泛素化酶，Hul5 是一种泛素连接酶，它们的作用与 彼此; Ubp6 将去除 Hul5 添加到底物上的泛素基团。两人均被招募 当 UPS 受损或受到挑战时，蛋白酶体会发挥作用。 Ubp6 的特异性非常显着，因为它只起作用 在携带多个泛素链的底物上。我们将进一步描述这种特殊性并研究其 机制基础。我们报道了 Hul5 在蛋白酶体上充当 E4——它使蛋白质泛素化 已经泛素化了。我们现在将重点关注此 E4 活动如何促进 蛋白酶体，因为 Hul5 似乎是持续过程的主要调节因子。我们将重构持续性效应 在纯化系统中，并在单分子分析中使用 Hul5 中的高度特异性突变体。我们也会调查 Ubp6 和 Hul5 受压力控制的机制。招募的第三个主要因素 处于蛋白稳态应激下的蛋白酶体是Ecm29，它具有同时结合RP和CP的独特功能。 Ecm29 很可能通过桥接这些复合物来调节蛋白酶体的活性和组装。 我们的研究将集中于 Ecm29 如何被招募到有缺陷的蛋白酶体以及它如何影响其结构和 稳定。最后，蛋白酶体的底物识别是由六种不同的泛素受体介导的。然而， 我们对酵母的详细遗传分析表明至少存在一种额外的未知泛素 蛋白酶体内的受体。我们将尝试识别这种受体，一旦我们产生了适当精确的 对于突变体，我们将探索该受体在底物识别和加工中的作用。总而言之，我们建议， 通过应用遗传学、酶学、结构生物学和定量全局蛋白质组学的方法， 阐明蛋白酶体功能和调节的主要途径。