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

• 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
• 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

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.

项目概要 蛋白质降解受到 AAA+ 家族的 ATP 依赖性区室蛋白酶的严格调控。这 真核细胞中主要的 AAA+ 蛋白酶是 26S 蛋白酶体，它是一种降解蛋白质的 35 个亚基复合物 带有多聚泛素链，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，但其底物选择的详细机制 和加工，特别是其调节和微调，例如通过底物附着的泛素 链，仍然基本上难以捉摸。在过去的资助期间，我们能够显着推进我们的 了解蛋白酶体的结构和功能。我们解决了基板的高分辨率结构- 在 ATP 水解循环的不同阶段参与蛋白酶体，建立了第一个完整的动力学 底物降解图片，揭示了蛋白酶体的主要构象变化是如何耦合的 到基板处理的各个步骤，并揭示了这些构象转变是如何部分地进行的 受蛋白酶体亚复合物之间的相互作用调节。我们的生化工具，重组表达 系统和位点特异性荧光标记策略使我们处于独特的地位来解决众多问题 关于泛素介导的蛋白质周转、26S 的分子机制的突出问题 蛋白酶体和其他 AAA+ 马达，以及与泛素蛋白酶体连接的途径的调节 系统。特别是我们新建立的基于单分子 FRET 的检测方法可以进行前所未有的研究 通过蛋白酶体调节颗粒的底物相互作用和进展，以及 蛋白酶体的构象动力学。令人兴奋的初步数据表明底物附着的泛素 链影响构象转换、底物接合和降解的动力学以及 蛋白酶体的展开能力取决于链长度和连接类型。主要目标是 研究蛋白酶体如何利用其三个主要泛素受体和之间的变构网络 蛋白酶体子复合物读出这个“泛素代码”并微调其活性。我们将聘请一名 多学科方法，包括体外生化、单分子和原子分辨率结构 研究。 26S 蛋白酶体上游的一条通路是 AAA+ 蛋白解折叠酶 Cdc48（p97/VCP 人类）。在一个新的研究方向中，我们将使用基于荧光和 FRET 的检测方法，并结合 一系列差异泛素化和标记的模型蛋白，以研究 Cdc48 如何与其复合 适配器 Ufd1/Npl4 接合并展开其底物，以及 Cdc48 适配器相互作用的动态 确定底物递送、解折叠和去泛素化。除了增进我们对 泛素依赖性蛋白质的展开和降解，我们的研究也具有重要的医学意义 26S蛋白酶体和26S蛋白酶体为新型小分子药物的开发提供了巨大的潜力 p97 在所有细胞中发挥多种调节功能，并在各种人类疾病中发挥重要作用。