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DEFINING THE FUNCTION OF PROTEASOMAL DEUBIQUITINASES

定义蛋白酶体去泛素酶的功能

基本信息

批准号：
10454952
负责人：
ERIC Robert STRIETER
金额：
$ 36.14万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10454952
关键词：
26S proteasome ATP-Dependent Proteases Address Affect Architecture Attention Binding Biological Biological Assay CRISPR/Cas technology Cell Line Cells Cellular biology Complex Crystallography Cyclic Peptides Data Deubiquitination Deuterium Development Disease Embryo Enzymes Failure Funding Generations Health Human Human Biology Hydrogen Kinetics Knowledge Label Light Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Modification Molecular Neurodegenerative Disorders Pathway interactions Peptide Hydrolases Phage Display Physiological Play Polymers Positioning Attribute Process Proteasome Binding Protein Chemistry Proteins Proteomics Research Role Signal Transduction Specificity Structure System Testing Translations Ubiquitin Work X-Ray Crystallography cellular targeting genome editing inhibitor innovation insight interdisciplinary approach kidney cell multicatalytic endopeptidase complex novel novel therapeutics premature prevent protein degradation proteostasis small molecule therapeutic target tool

项目摘要

Project Summary The 26S proteasome is a massive, intricately regulated ATP-dependent protease that is responsible for the degradation of most cellular proteins. Failure of the proteasome to precisely regulate protein levels is a hallmark of many cancers and neurodegenerative diseases. Targeting proteins to the proteasome requires covalent modification with a polymeric (Ub) chain. Thus, it is perplexing that the proteasome actually houses enzymes (deubiquitinases/DUBs) responsible for removing Ub chains. While it has become clear that the intrinsic DUB, RPN11, promotes degrades by preventing premature deubiquitination of proteins not yet engaged with the proteasome, the roles of other proteasomal DUBs, e.g., UCH37/UCHL5, are poorly understood. In preliminary studies, we discovered that proteasome-bound UCH37 acts as a chain editor by removing branch points. This application proposes to elucidate how UCH37 selects branched Ub chains for editing, how this activity is integrated into the entire process of proteasomal degradation, and how it impacts the turnover of cellular proteins. In Aim 1, we will investigate the role of UCH37-mediated chain debranching during proteasomal degradation using distinct, purified human proteasome complexes and fluorescent, polyubiquitinated substrates. In Aim 2, we propose to identify cellular targets of proteasome-bound UCH37 using an innovative combination of quantitative proteomics, in-cell proximity labeling, and Ub middle-down mass spectrometry. In Aim 3, we focus on understanding the molecular basis of UCH37's specificity toward branched chains. Finally, in Aim 4, we will develop novel cyclic peptide inhibitors of UCH37 to facilitate efforts to dissect its function in any biological paradigm. The knowledge gained from this research will shed light on fundamental aspects of the ubiquitin proteasome system and pave the way for the development of new therapeutics that regulate proteasome function.

项目概要 26S 蛋白酶体是一种巨大的、复杂调节的 ATP 依赖性蛋白酶，负责大多数细胞蛋白质的降解。蛋白酶体无法精确调节蛋白质水平是一个原因许多癌症和神经退行性疾病的标志。将蛋白质靶向蛋白酶体需要用聚合 (Ub) 链进行共价修饰。因此，令人困惑的是，蛋白酶体实际上容纳了负责去除 Ub 链的酶（去泛素酶/DUB）。虽然已经明确的是内在 DUB，RPN11，通过防止尚未发生的蛋白质过早去泛素化来促进降解与蛋白酶体结合后，其他蛋白酶体 DUB（例如 UCH37/UCHL5）的作用很差明白了。在初步研究中，我们发现蛋白酶体结合的 UCH37 通过以下方式充当链编辑器：删除分支点。本申请旨在阐明 UCH37 如何选择支化 Ub 链编辑，该活动如何整合到蛋白酶体降解的整个过程中，以及它如何影响细胞蛋白质的周转。在目标 1 中，我们将研究 UCH37 介导的链脱支的作用在蛋白酶体降解过程中，使用独特的、纯化的人类蛋白酶体复合物和荧光，多泛素化底物。在目标 2 中，我们建议鉴定蛋白酶体结合的 UCH37 的细胞靶标使用定量蛋白质组学、细胞内邻近标记和 Ub 中下的创新组合质谱分析。在目标 3 中，我们重点了解 UCH37 特异性的分子基础支链。最后，在目标4中，我们将开发UCH37的新型环肽抑制剂以促进努力剖析其在任何生物学范式中的功能。从这项研究中获得的知识将揭示泛素蛋白酶体系统的基本方面，并为新的开发铺平道路调节蛋白酶体功能的疗法。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ubiquitination drives COPI priming and Golgi SNARE localization.

DOI：
10.7554/elife.80911
发表时间：
2022-07-29
期刊：
ELIFE
影响因子：
7.7
作者：
Date, Swapneeta S.;Xu, Peng;Hepowit, Nathaniel L.;Diab, Nicholas S.;Best, Jordan;Xie, Boyang;Du, Jiale;Strieter, Eric R.;Jackson, Lauren P.;MacGurn, Jason A.;Graham, Todd R.
通讯作者：
Graham, Todd R.

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains.