Structural and Functional Analysis of Proteasome Core Particle Biogenesis

蛋白酶体核心颗粒生物发生的结构和功能分析

基本信息

批准号：
10609420
负责人：
John W Hanna
金额：
$ 40.73万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-15 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10609420
关键词：
26S proteasome Active Sites Affinity Affinity Chromatography Alzheimer&apos s Disease Architecture Atlases Binding Biogenesis Biology Cells Complex Coupled Cryoelectron Microscopy Data Dedications Disease Eukaryota Failure Generations Genetic Transcription Goals Light Malignant Neoplasms Mediating Modeling Molecular Chaperones Multiple Myeloma N-terminal Nature Neurodegenerative Disorders Nucleosome Core Particle Pathway interactions Peptide Hydrolases Peptides Physiological Positioning Attribute Process Productivity Proteasome Binding Proteasome Inhibition Proteins Proteolysis Regulon Resolution Role Shapes Stress Structure Testing Therapeutic Time Ubiquitin Work Yeasts cancer therapy cell type human disease in vitro testing in vivo inhibitor innovation insight interest member misfolded protein multicatalytic endopeptidase complex mutant novel novel therapeutic intervention particle pharmacologic protein degradation protein misfolding proteotoxicity response

项目摘要

Project Summary/Abstract Protein misfolding is a key feature of many human diseases including most neurodegenerative diseases and many cancers. Destruction of misfolded proteins is largely mediated by the proteasome, a 2.5 MDa multisubunit complex which is the most sophisticated protease ever described. The proteasome's active sites are sequestered within a barrel-shaped cylindrical chamber, known as the core particle (CP). Access of substrates to the CP is mediated by the regulatory particle (RP), which recognizes proteasome substrates via their ubiquitin tags. The RP unfolds, deubiquitinates, and injects the substrate into the CP where it is rendered into small peptides. Pharmacologic inhibition of the proteasome is an established anti-cancer therapy, most notably in multiple myeloma. Conversely, the possibility of enhancing proteasome function has generated considerable interest in recent years. Such a strategy might ameliorate diseases caused by protein misfolding. A key step in the generation of active proteasomes is the assembly of the 700 kDa 28-subunit CP, which precedes assembly of the full proteasome and occurs by an ordered multistep pathway that requires the function of five dedicated chaperone proteins. Structural analysis of CP maturation has been hampered by challenges in isolating and characterizing assembly intermediates due to their low abundance and transitory nature. Here we hypothesized that defined CP mutants may be enriched for assembly intermediates. We have developed a productive work-flow for the affinity purification and structural analysis of these mutants, and have already generated eight high resolution structures. In Aim 1, we will carry out this structural analysis of CP mutants using Cryo-Electron Microscopy, coupled with detailed structure-function analyses. In Aim 2, we will characterize a long-known but poorly understood regulator of the CP known as PI31/Fub1. We will attempt to determine its structure in complex with the CP using Cryo-Electron Microscopy, and test a number of specific hypotheses regarding its function. In Aim 3, we will characterize a novel protein which is a previously unrecognized transcriptional target of the Rpn4-mediated proteasome biogenesis regulon, and which appears to be a new proteasome-interacting protein. This proposal is expected to provide significant insight into proteasome assembly and overall function, information which could lead to novel therapeutic strategies based on modulating proteasome activity to treat diseases characterized by protein misfolding.

项目概要/摘要蛋白质错误折叠是许多人类疾病（包括大多数神经退行性疾病）的一个关键特征和许多癌症。错误折叠蛋白质的破坏主要是由蛋白酶体介导的，蛋白酶体是一种 2.5 MDa 多亚基复合物是迄今为止描述的最复杂的蛋白酶。蛋白酶体的活性位点被隔离在桶形圆柱形室内，称为核心颗粒（CP）。访问 CP 的底物是由调节颗粒 (RP) 介导的，RP 可以通过以下方式识别蛋白酶体底物：他们的泛素标签。 RP 展开、去泛素化，并将底物注入 CP 中进行渲染成小肽。蛋白酶体的药物抑制是一种既定的抗癌疗法，大多数尤其是多发性骨髓瘤。相反，增强蛋白酶体功能的可能性已经产生近年来引起了极大的兴趣。这种策略可能会改善由蛋白质错误折叠引起的疾病。产生活性蛋白酶体的关键步骤是组装 700 kDa 28 亚基 CP，它在完整蛋白酶体组装之前，通过需要该功能的有序多步骤途径发生五种专用伴侣蛋白。 CP 成熟的结构分析受到以下挑战的阻碍：由于组装中间体的丰度低且短暂，因此可以对其进行分离和表征。在这里我们假设定义的 CP 突变体可能会富集组装中间体。我们开发了一个用于这些突变体的亲和纯化和结构分析的高效工作流程，并且已经生成了八个高分辨率结构。在目标 1 中，我们将使用以下方法对 CP 突变体进行结构分析冷冻电子显微镜，加上详细的结构功能分析。在目标 2 中，我们将描述众所周知但知之甚少的 CP 调节因子 PI31/Fub1。我们将尝试确定其使用冷冻电子显微镜研究与 CP 的复合物结构，并测试一些特定的假设关于它的功能。在目标 3 中，我们将表征一种以前未被识别的新型蛋白质 Rpn4 介导的蛋白酶体生物合成调节子的转录靶标，这似乎是一个新的蛋白酶体相互作用蛋白。该提案预计将为蛋白酶体提供重要的见解组装和整体功能，这些信息可能导致基于调节的新治疗策略蛋白酶体活性可治疗以蛋白质错误折叠为特征的疾病。