ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

真核蛋白酶体的 ATP 依赖性蛋白质解折叠和易位

基本信息

批准号：
10298469
负责人：
Andreas Martin
金额：
$ 31.29万
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 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298469
关键词：
26S proteasome ATP Hydrolysis Address Affect Binding Biochemical Biochemistry Biological Assay Cell Survival Cell division Cells Characteristics Code Complement Complex Coupled Cryoelectron Microscopy Data Deubiquitination Development Dissection Electron Microscopy Engineering Eukaryotic Cell Family Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Fluorescent Dyes Genetic Transcription Goals Grant Human In Vitro Individual Intervention Kinetics Label Length Malignant Neoplasms Measurement Mechanics Mediating Medical Modeling Modification Molecular Molecular Conformation Molecular Machines Motor Mutation Neurodegenerative Disorders Pathway interactions Peptide Hydrolases Pharmaceutical Preparations Pharmacologic Substance Play Polyubiquitin Positioning Attribute Process Protac Protein Engineering Proteins Quality Control Recombinants Regulation Research Resolution Role Series Site Structure Substrate Interaction System Ubiquitin Work Yeasts base biochemical tools biological adaptation to stress conformational conversion encryption human disease insight interdisciplinary approach multicatalytic endopeptidase complex particle protein degradation protein folding proteostasis receptor reconstitution single molecule single-molecule FRET small molecule small molecule inhibitor tool translocase unfoldase unnatural amino acids valosin-containing protein

项目摘要

Project Summary Protein degradation is tightly regulated by ATP-dependent compartmental proteases of the AAA+ family. The major AAA+ protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and controls protein homeostasis as well as numerous vital processes. Despite the proteasome’s great importance for cell viability, its detailed mechanisms for substrate selection and processing, and in particular its regulation and fine-tuning, for instance by substrate-attached ubiquitin chains, remain largely elusive. During the past granting period, we were able to significantly advance our understanding of proteasome structure and function. We solved high-resolution structures of the substrate- engaged proteasome at different stages of the ATP-hydrolysis cycle, established the first complete kinetic picture of substrate degradation, revealed how major conformational changes of the proteasome are coupled to individual steps of substrate processing, and uncovered how these conformational transitions are in part regulated by interactions between proteasomal subcomplexes. Our biochemical tools, recombinant expression systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S proteasome and other AAA+ motors, and the regulation of pathways connected to the ubiquitin-proteasome system. Especially our newly established single-molecule FRET-based assays allow unprecedented studies of substrate interactions and progression through the proteasome regulatory particle, as well as the conformational dynamics of the proteasome. Exciting preliminary data indicate that substrate-attached ubiquitin chains affect the conformational switching, the kinetics of substrate engagement and degradation, and the unfolding power of the proteasome depending on the chain length and linkage type. A primary goal is to investigate how the proteasome utilizes its three main ubiquitin receptors and allosteric networks between proteasomal subcomplexes to read out this “ubiquitin code” and fine-tune its activities. We will employ a multidisciplinary approach that includes in-vitro biochemical, single-molecule, and atomic-resolution structural studies. A pathway upstream of the 26S proteasome is the AAA+ protein unfoldase Cdc48 (p97/VCP in human). In a new research direction, we will use fluorescence- and FRET-based assays combined with a series of differentially ubiquitinated and labeled model proteins to investigate how Cdc48 in complex with its adaptor Ufd1/Npl4 engages and unfolds its substrates, and how the dynamics of Cdc48-adaptor interactions determine substrate delivery, unfolding, and deubiquitination. Besides advancing our general understanding of ubiquitin-dependent protein unfolding and degradation, our research also has substantial medical relevance and offers great potential for the development of new small-molecule drugs, as both the 26S proteasome and p97 fulfill numerous regulatory functions in all cells and play important roles in various human diseases.

项目摘要蛋白质降解是通过AAA+家族的ATP依赖性隔室蛋白酶紧密的真核细胞中主要的AAA+整体是26S蛋白酶体，这是一种降解蛋白质的35个亚基复合物用多泛素链标记并控制蛋白质稳态以及许多重要过程。尽管蛋白酶体对细胞活力非常重要，但它详细介绍了底物选择的机制和处理，尤其是其法规和微调，例如附属的泛滥连锁店，在过去的授予期内仍然难以捉摸了解蛋白酶体的结构和功能。在ATP-HYDRORYSISIS周期的不同阶段参与蛋白酶体，建立了第一个完整动力学底物退化的图片，陶醉以底物处理的各个步骤通过蛋白酶体亚复合物之间的相互作用。系统和特定地点的荧光标签策略使我们处于独特的位置，以应对众多关于泛素介导的蛋白质周转率，26S的分子机制的杰出问题蛋白酶体和其他AAA+电动机，以及连接到泛素 - 蛋白酶体的途径的调节系统。底物相互作用和通过蛋白酶体调节粒子的进展以及蛋白酶体的构象动力学。链影响授权转换，底物参与和降解的动力学以及你蛋白酶体的展开力量取决于链长和连锁类型。研究蛋白酶体如何利用其ITILILITIL，将其三种主要的泛素受体和变构网络之间我们将雇用一个。多次攻击是体外生化，单分子和原子分辨率结构研究。人）。一系列差异化泛素化和标记的模型蛋白，以研究CDC48如何与ITS一起进行适配器UFD1/NPL4参与并展开其底物，以及Cdc48-Apaptor的动态如何相互作用确定底物递送，展开和去泛素化。我们的研究也具有很大的医学相关性并为开发新的小分子药物提供了巨大的潜力，包括26S蛋白酶体和 p97履行了所有细胞中的众多调节功能，并在各种人类疾病中起重要作用。