ATP-dependent protein unfolding and translocation by the eukaryotic proteasome

真核蛋白酶体的 ATP 依赖性蛋白质展开和易位

基本信息

批准号：
8186314
负责人：
Andreas Martin
金额：
$ 24.1万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8186314
关键词：
26S proteasome ATP Hydrolysis ATP phosphohydrolase ATP-Dependent Proteases Apoptosis Area Binding Biochemical Biochemistry Biological Biological Models Biomedical Research Biophysics Cell Cycle Regulation Cell Differentiation process Cells Chemicals Communication Complex Coupled Coupling Data Development Disease Drug Delivery Systems Elements Enzymes Escherichia coli Eukaryotic Cell Event Family Generations Genetic Transcription Goals Grant Homeostasis Homo In Vitro Individual Insecta Intervention Kinetics Knowledge Maintenance Malignant Neoplasms Mechanics Methods Molecular Molecular Biology Molecular Machines Mutagenesis Pathogenesis Pathway interactions Peptide Hydrolases Pharmacologic Substance Polyubiquitin Process Prokaryotic Cells Protein Biochemistry Proteins Regulation Research Role Signal Transduction Speed Structure Substrate Interaction System Time Training Translating Ubiquitin Ubiquitination Work base biological research experience genetic regulatory protein human disease in vivo interest laser tweezer mechanical drive member multicatalytic endopeptidase complex novel optical traps polypeptide protein degradation reconstitution research study single molecule tool unfoldase

项目摘要

DESCRIPTION (provided by applicant): Degradation of proteins is highly specific and tightly regulated by energy-dependent compartmental proteases in all prokaryotic and eukaryotic cells. These enzymes, members of the AAA+ ATPase family, use ATP hydrolysis to drive the mechanical unfolding of protein substrates and their translocation into a sequestered degradation chamber. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, which controls protein homeostasis and numerous vital processes by specifically degrading regulatory proteins involved for instance in transcription, cell-cycle control, signal transduction, and apoptosis. Most proteasomal substrates are marked for degradation by the reversible attachment of a poly-ubiquitin chain, which acts as a tethering signal for substrate delivery. Substantial knowledge about ubiquitin-tagging and de-ubiquitinating systems is already available, but only very little is known about the detailed mechanisms that control substrate degradation by the proteasome. The long-term objective of this proposal is to understand the molecular bases for substrate recognition, ATP-dependent forceful unfolding and translocation, and the regulation thereof by de- ubiquitination and fine-tuning of the proteasomal unfolding machinery. My lab has devised novel systems for the heterologous expression of the proteasomal 19S base in E.coli and insect cells, and the reconstitution of functional 26S proteasomes in vitro. This provides us with powerful tools for extensive mutagenesis and unprecedented mechanistic studies. Using a combination of biochemical and biophysical approaches, our goals are to 1) further develop the eukaryotic 26S proteasome for quantitative in-vitro analyses, 2) determine the molecular mechanisms underlying coordinated ATP-hydrolysis, substrate interactions, and the timing of de-ubiquitination, and 3) understand the mechano-chemical coupling and the generation of unfolding force. We anticipate that our results will contribute to the general understanding of ATP-dependent molecular machines, ubiquitin signaling, and the regulation of protein turnover in eukaryotic cells, and thus impact several different areas of biochemistry, molecular biology, and cell-biological research. Given the role of the proteasome in the pathogenesis of numerous human diseases, a detailed knowledge of the molecular mechanisms for substrate processing has also significant biomedical relevance and may aid the development of novel, more specific drugs targeting the 26S proteasome. PUBLIC HEALTH RELEVANCE: The eukaryotic 26S proteasome is a proteolytic molecular machine that regulates many vital processes in the cell. This proposal aims to understand in molecular detail the biochemistry, mechanics, and regulation of substrate recognition, ATP-dependent unfolding, and processing by the proteasome.

描述（由申请人提供）：蛋白质的降解具有高度特异性，并受到所有原核和真核细胞中能量依赖性区室蛋白酶的严格调节。这些酶是 AAA+ ATP 酶家族的成员，利用 ATP 水解来驱动蛋白质底物的机械展开及其易位到隔离的降解室中。真核细胞中主要的 ATP 依赖性蛋白酶是 26S 蛋白酶体，它通过特异性降解参与转录、细胞周期控制、信号转导和细胞凋亡等的调节蛋白来控制蛋白质稳态和许多重要过程。大多数蛋白酶体底物通过多聚泛素链的可逆附着而被标记为降解，该链充当底物递送的束缚信号。关于泛素标记和去泛素化系统的大量知识已经存在，但对蛋白酶体控制底物降解的详细机制知之甚少。该提案的长期目标是了解底物识别、ATP 依赖性强力解折叠和易位的分子基础，以及通过蛋白酶体解折叠机制的去泛素化和微调对其进行调节。我的实验室设计了新的系统，用于在大肠杆菌和昆虫细胞中异源表达蛋白酶体 19S 碱基，并在体外重建功能性 26S 蛋白酶体。这为我们提供了进行广泛诱变和前所未有的机制研究的强大工具。结合生物化学和生物物理方法，我们的目标是 1) 进一步开发用于定量体外分析的真核 26S 蛋白酶体，2) 确定协调 ATP 水解、底物相互作用和去泛素化时间的分子机制，以及 3）了解机械化学耦合和展开力的产生。我们预计我们的结果将有助于对 ATP 依赖性分子机器、泛素信号传导和真核细胞中蛋白质周转调节的一般理解，从而影响生物化学、分子生物学和细胞生物学研究的几个不同领域。鉴于蛋白酶体在多种人类疾病发病机制中的作用，对底物加工分子机制的详细了解也具有重要的生物医学相关性，并可能有助于开发针对 26S 蛋白酶体的新型、更具特异性的药物。公共卫生相关性：真核 26S 蛋白酶体是一种蛋白水解分子机器，可调节细胞中的许多重要过程。该提案旨在从分子细节上了解底物识别、ATP 依赖性展开和蛋白酶体加工的生物化学、力学和调节。