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.

项目摘要/摘要 我实验室的工作重点是蛋白酶体，这是一种降解泛素蛋白的蛋白酶 共轭关系。已知蛋白酶体是泛素-蛋白酶体系统(UPS)的中心活性 从UPS被绘制出来的时候起，但它的重要性在当时并没有得到充分的认识，因为 蛋白酶体被错误地认为是不受调控的和被动的--本质上是一种哑酶。但这件事 人们对精细监管的不同机制的逐步认同颠覆了人们的看法 蛋白酶体的合成、周转、定位、底物专一性和专一性。这些机制是 具有特殊的意义，因为它们提供了对UPS输出进行全球控制的手段。同时，有证据表明 蛋白酶体活性在疾病中的重要性已经积累起来。例如，即使是适度提升的 蛋白酶体水平大大延长了黑腹隐翅虫和线虫的寿命，并提高了它们的能力 以抵御压力，如有毒的多聚Q蛋白的表达。一种迷人的蛋白酶体模式 调节是涉及蛋白酶体底物上泛素链的动态重新配置。二 这种链编辑因子在真核生物中高度活跃和保守：Ubp6/USP14和 Hul5/UBE3C。Ubp6是一种去泛素化酶，Hul5是一种泛素连接酶，它们的作用是相反的 Ubp6将去除由Hul5添加到底物上的泛素基团。两人都被招募到 当UPS受损或受到挑战时，蛋白酶体。Ubp6的独特之处在于它只起作用 在携带多条泛素链的底物上。我们将进一步描述这一特性，并研究其 机械论基础。我们已经报道了Hul5在蛋白酶体上的功能是E4-它泛素化蛋白质 它们已经无处不在。我们现在将重点关注E4活动如何促进 蛋白酶体，因为Hul5似乎是加工能力的主要调节因子。我们将重建过程性效应 在纯化的系统中，并在单分子分析中使用Hul5中高度特异的突变体。我们还将调查 Ubp6和Hul5受胁迫控制的机制。招聘的第三个主要因素是 蛋白稳定胁迫下的蛋白酶体为Ecm29，它具有结合RP和CP的独特特性。 ECM29调节蛋白酶体的活性和组装，很可能是通过桥接这些复合体。 我们的研究将集中在ECM29如何被招募到有缺陷的蛋白酶体上，以及它如何影响它们的结构和 稳定性。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， 我们在酵母中的详细基因分析表明，至少存在另外一种未知的泛素 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 突变体，我们将探索该受体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。