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.

项目摘要 26S蛋白酶体是一种庞大的，复杂调节的ATP依赖性蛋白酶，负责大多数细胞蛋白的降解。蛋白酶体的失败精确调节蛋白质水平是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与聚合物（UB）链的共价修饰。因此，令人困惑的是，蛋白酶体实际上是容纳负责去除UB链的酶（去泛素酶/配音）。虽然已经很清楚内在的配音RPN11通过防止蛋白质过早去泛素化来促进降解参与蛋白酶体，其他蛋白酶体配音的作用，例如UCH37/UCHL5，很差理解。在初步研究中，我们发现蛋白酶体结合的UCH37通过删除分支点。该应用程序建议阐明UCH37如何选择分支UB链编辑，该活动如何集成到蛋白酶体降解的整个过程中，以及它如何影响细胞蛋白的营业额。在AIM 1中，我们将调查UCH37介导的链分支的作用在蛋白酶体降解过程中，使用不同的，纯化的人蛋白酶体复合物和荧光，多泛素化的底物。在AIM 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，内部接近标签和UB中间的创新组合质谱法。在AIM 3中，我们专注于理解UCH37特异性的分子基础分支链。最后，在AIM 4中，我们将开发新型的UCH37的环状肽抑制剂，以促进努力在任何生物范式中剖析其功能。从这项研究中获得的知识将阐明泛素蛋白酶体系统的基本方面，并为新的发展铺平了道路调节蛋白酶体功能的治疗剂。

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.

Quantitative Analysis of Diubiquitin Isomers Using Ion Mobility Mass Spectrometry.

使用离子淌度质谱法定量分析双泛素异构体。