Regulation of Proteasome Activity

蛋白酶体活性的调节

• 批准号: 10707061
• 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/10707061
• 关键词: Affect; 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; model organism; multicatalytic endopeptidase complex; mutant; polyglutamine; proteostasis; receptor; reconstitution; recruit; single molecule; structural biology; ubiquitin isopeptidase; 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.

项目摘要 /摘要 我实验室努力的重点是蛋白酶体，蛋白酶会降解泛素 - 蛋白质 共轭。蛋白酶体是泛素 - 蛋白酶体系统（UPS）中的中心活动 从UPS绘制的时间开始，但当时它的意义尚未得到完全认可，因为 蛋白酶体是错误地提出的，不受监管和被动地提出了一种愚蠢的酶。但这 通过对细节调节的潜水机制的逐步识别，人们已经推翻了感知 蛋白酶综合，周转，定位，底物特异性和特定活性。这些机制是 特别感兴趣，因为它们为全球控制UPS输出提供了手段。同时，证据 蛋白酶体活性在疾病中的重要性已积累。例如，即使是适度的海拔 蛋白酶体水平大大提高了梅拉诺斯特和秀丽隐杆线虫的寿命，以及它们的能力 承受诸如有毒Polyq蛋白的表达之类的应力。一种迷人的蛋白酶体模式 调节是涉及泛素链在蛋白酶体处的底物上的动态重新配置。二 这种链条编辑的因素高度活跃，并且在整个真核生物中保持了保守：UBP6/USP14和 HUL5/UBE3C。 UBP6是一种去泛素化酶，HUL5是一种泛素连接酶，它们与反对 彼此; UBP6将通过HUL5删除添加到底物中的泛素组。两者都被招募到 当UPS受到损害或挑战时的蛋白酶体。 UBP6的特异性非常出色，因为它仅行动 在携带多个泛素链的底物上。我们将进一步表征这种特异性并研究其 机械基础。我们报告说，HUL5在蛋白质组上充当E4 - 它泛素蛋白质 已经泛素化了。现在，我们将重点关注E4活动如何促进 蛋白酶体，因为HUL5似乎是加工性的主要调节剂。我们将重建过程效应 在纯化的系统中，在单分子分析中使用HUL5中的高度特异性突变体。我们还将调查 UBP6和HUL5由应力控制的机制。招募的第三个主要因素 蛋白质抗压胁迫下的蛋白酶体是ECM29，其具有结合RP和CP的独特特征。 ECM29调节蛋白酶体的活性和组装，最有可能通过桥接这些复合物来调节。 我们的研究将重点介绍如何将ECM29招募到错误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个不同的泛素接收器介导的。然而， 我们在酵母中的详细遗传分析表明存在至少一个额外的未知泛素 蛋白酶体内的受体。我们将尝试识别该受体，一旦我们产生了适当的精确 突变体我们将探讨该接收器在底物识别和处理中的作用。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球蛋白质组学的方法 阐明蛋白酶体功能和调节的主要途径。