Mechanism of gate-opening in the 20S proteasome induced by the proteasomal ATPase

蛋白酶体ATP酶诱导20S蛋白酶体开门的机制

基本信息

批准号：
9037029
负责人：
Yifan Cheng
金额：
$ 26.78万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9037029
关键词：
26S proteasome ATP Hydrolysis ATP phosphohydrolase Active Sites Address Allosteric Regulation Amino Acids Architecture Binding Biology Cell physiology Cells Communication Complex Cryoelectron Microscopy Data Elements Ensure Eukaryota Eukaryotic Cell Health Huntington Disease Immune system In Vitro Individual Knowledge Life Malignant Neoplasms Mediating Methods Modeling Molecular Biology Molecular Chaperones Molecular Conformation Molecular Machines Mutate Mutation Names Neurodegenerative Disorders Nucleosome Core Particle Nucleotides Pathogenesis Pathway interactions Peptides Play Process Proteins Recombinants Regulation Resolution Role Shapes Signaling Protein Site Structure Testing Ubiquitin Yeasts base biochemical tools biophysical tools dimer human disease in vivo multicatalytic endopeptidase complex novel strategies particle prevent protein degradation reconstitution reconstruction tool

项目摘要

DESCRIPTION (provided by applicant): In eukaryotes the ATP dependent protein degradation by the ubiquitin-proteasome pathway removes short lived signaling protein that is critical in regulation of cellular process, degrades misfolded and damaged proteins whose accumulation is toxic to the cell and breaks down foreign proteins to generate antigenic peptides for presenting to the immune system. It is fundamental in understanding the mechanism of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington disease. The eukaryotic 26S proteasome is formed by a 20S proteasome with the proteolytic active sites sequestered inside it and two 19S regulatory particles each contain six ATPases in contact with the 20S. A key role of the ATPases is to open the gated channel in the 20S to facilitate substrates enter for destruction. An important question in proteasome biology is that how short peptides of proteolytic products are released efficiently from CP to ensure a continuous substrate entering and products release required for the degradation of large protein substrates. A widely accepted yet untested paradigm is that the 26S proteasome functions unidirectional in which unfolded substrates enter the CP from one end and the proteolytic products exit from the opposite end. Another important question is what is the role of ATP hydrolysis by Rpt subunits during the Rpt ring assembly, and if the assembly requires CP as a template? In this application, we aim to address these questions. We will use near atomic resolution single particle cryoEM as our main structural analysis tool, together with other methods in molecular biology, biochemistry and biophysical tool, to elucidate the mechanisms that regulates the asymmetrical functionality of the symmetrical protein degradation machinery. The specific aims are (1) determine the mechanism of proteolytic products releasing from the 20S degradation chamber, (2) determine mechanism that coordinates the functions of proteasomal activators bound to the opposite ends of 20S core particle, and (3) determine the role of ATP hydrolysis in the assembly pathway of eukaryotic proteasomal ATPases. Substantial completion of these aims will advance our knowledge about the proteasome-mediated protein degradation that plays a key role in the pathogenesis of many human diseases.

描述（由申请人提供）：在真核生物中，泛素-蛋白酶体途径的 ATP 依赖性蛋白质降解去除了在细胞过程调节中至关重要的短命信号蛋白，降解了错误折叠和受损的蛋白质，这些蛋白质的积累对细胞有毒，并分解了外源蛋白。蛋白质产生抗原肽以呈递给免疫系统。它对于理解许多人类疾病的机制至关重要，特别是癌症和神经退行性疾病，例如神经退行性疾病。亨廷顿病。真核26S蛋白酶体由20S蛋白酶体形成，其中蛋白水解活性位点被隔离在其中，两个19S调节颗粒各自含有六个与20S接触的ATP酶。 ATP 酶的一个关键作用是在 20 年代打开门控通道，以促进底物进入并被破坏。蛋白酶体生物学中的一个重要问题是蛋白水解产物的短肽如何从CP中有效释放，以确保大蛋白底物降解所需的底物连续进入和产物释放。一种广泛接受但未经测试的范例是 26S 蛋白酶体单向发挥作用，其中未折叠的底物从一端进入 CP，而蛋白水解产物从另一端退出。另一个重要问题是Rpt环组装过程中Rpt亚基水解ATP的作用是什么，以及组装是否需要CP作为模板？在此应用程序中，我们旨在解决这些问题。我们将使用近原子分辨率的单粒子冷冻电镜作为我们的主要结构分析工具，结合分子生物学、生物化学和生物物理工具中的其他方法，来阐明调节对称蛋白质降解机制的不对称功能的机制。具体目标是（1）确定从 20S 降解室释放蛋白水解产物的机制，（2）确定协调结合到 20S 核心颗粒两端的蛋白酶体激活剂功能的机制，以及（3）确定真核蛋白酶体 ATP 酶组装途径中的 ATP 水解。这些目标的实质性完成将增进我们对蛋白酶体介导的蛋白质降解的了解，这种降解在许多人类疾病的发病机制中发挥着关键作用。