Dissecting the unique chaperone mechanism of the Rvb1/Rvb2 AAA+ ATPase complex

剖析 Rvb1/Rvb2 AAA ATPase 复合物的独特伴侣机制

• 批准号: 10395443
• 负责人: Elise Noelle Muñoz
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10395443
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Acute Myelocytic Leukemia; Adopted; Architecture; Biochemical; Biological Process; Biophysics; Cell physiology; Cells; Cellular Stress; Chromatin; Chromatin Remodeling Factor; Complex; Coupled; Cryoelectron Microscopy; Cystic Kidney Diseases; DNA; DNA Damage; DNA biosynthesis; Defect; Disease; Exhibits; Family; Genetic Transcription; Goals; Histones; In Vitro; Laboratories; Lead; Malignant Neoplasms; Mediating; Modeling; Molecular Chaperones; Molecular Conformation; Molecular Structure; Motor; Nucleosomes; Pathway interactions; Physiological Processes; Primary carcinoma of the liver cells; Process; Protein Subunits; Proteins; Quality Control; Reaction; Research; Research Training; Resources; Role; Saccharomyces cerevisiae; Signal Transduction; Slide; Small Nucleolar Ribonucleoproteins; Structure; Supervision; Techniques; Testing; Time; Translations; Work; base; biochemical tools; chromatin remodeling; dimer; experimental study; helicase; in vivo; insight; prevent; protein complex; scaffold; tool

PROJECT SUMMARY/ABSTRACT Multi-subunit protein machines are fundamental to nearly all biological processes, ranging from DNA replication to protein translation. Assembly of these protein machines requires processes that enable coordination of necessary interactions between subunits while preventing inappropriate ones. One way the cell manages to do this is by utilizing chaperones, a diverse group of proteins that assist in the non-covalent folding and unfolding, and assembly and disassembly of macromolecular structures. Rvb1 and Rvb2 are two essential AAA+ ATPases from S. cerevisiae that are proposed to act together (Rvb1/Rvb2) as an assembly chaperone for various multi- subunit complexes, including select chromatin remodelers. Rvb1 and Rvb2 are also associated with various cellular processes, including transcription, DNA-damage, and cell signaling, and their defects are strongly associated with multiple cancers, including hepatocellular carcinoma and acute myeloid leukemia. Yet how the Rvb1/Rvb2 complex functions as a chaperone is poorly understood. This proposal will explore Rvb1/Rvb2 function in the context of assembly and activity of the INO80 and SWR1 chromatin remodeling complexes. INO80 and SWR1 are chromatin remodelers composed of multiple subunits, including a Rvb1/Rvb2 hexamer, that use the energy from ATP hydrolysis to remodel nucleosome substrates. There is evidence for Rvb1/Rvb2 chaperone-like activity in the context of INO80 complex assembly. A proposed intermediate of INO80 assembly contains Rvb1/Rvb2 and exhibits enhanced Rvb1/Rvb2 ATPase activity. However, Rvb1/Rvb2’s role throughout complex assembly remains unclear. INO80 and SWR1 have similar overall architectures, but have unique subunits and nucleosome remodeling activities. Distinct interactions between Rvb1/Rvb2 and the insertion regions of a core subunit, Ino80p and Swr1p, may regulate unique complex assembly and activity of INO80 and SWR1, respectively. This proposal will (i) identify and characterize intermediates of the Rvb1/Rvb2-mediated INO80 assembly pathway and (ii) test whether and how Ino80p and Swr1p insertion regions regulate specific complex assembly and activity. This will be done using a combination of in vitro biophysical and biochemical techniques. The results will uncover the steps in INO80 and SWR1 assembly that are regulated by Rvb1/Rvb2 and in doing so identify major points of quality control in the assembly and function of these complexes in vivo. The principles derived from this study will be broadly applicable to understanding how the Rvb1/Rvb2 complex functions in other cellular processes and how defective Rvb1/Rvb2 functioning contributes to specific diseases. This research will be performed at UCSF under the direct supervision of Dr. Geeta Narlikar, an expert in using biochemical tools to dissect mechanism of chromatin regulators. The Narlikar lab and UCSF is equipped with all tools and resources necessary to accomplish the proposed research and training goals.

项目摘要/摘要 多亚基蛋白质机器是几乎所有生物过程的基础，从DNA复制 蛋白质翻译。这些蛋白质机器的组装需要能够协调 子单位之间的必要的相互作用，同时防止不适当的。一种方法是 这是通过利用分子伴侣，一组帮助非共价折叠和解折叠的不同蛋白质， 以及大分子结构的组装和拆卸。Rvb 1和Rvb 2是两种必需的AAA+ ATP酶 来自酿脓链球菌酿酒酵母，提出一起行动（Rvb 1/Rvb 2）作为组装分子伴侣的各种多- 亚基复合物，包括选择染色质重塑。Rvb 1和Rvb 2还与各种不同的 细胞过程，包括转录、DNA损伤和细胞信号传导，以及它们的缺陷， 与多种癌症相关，包括肝细胞癌和急性髓性白血病。然而， Rvb 1/Rvb 2复合物作为分子伴侣的功能知之甚少。本提案将探讨Rvb 1/Rvb 2 在INO 80和SWR 1染色质重塑的组装和活性背景下的功能 配合物INO 80和SWR 1是由多个亚基组成的染色质重塑蛋白，包括Rvb 1/Rvb 2 六聚体，其使用来自ATP水解的能量来重塑核小体底物。有证据表明 在INO 80复合物组装的背景下，Rvb 1/Rvb 2分子伴侣样活性。INO 80的拟定中间体 组装体含有Rvb 1/Rvb 2，并表现出增强的Rvb 1/Rvb 2 ATP酶活性。Rvb 1/Rvb 2的作用 整个复杂的装配过程仍不清楚。INO 80和SWR 1具有类似的整体架构，但 独特的亚基和核小体重塑活性。Rvb 1/Rvb 2与 核心亚基Ino 80 p和Swr 1 p的插入区域可以调节独特的复合物组装和活性， INO 80和SWR 1。该提案将（i）确定和表征 Rvb 1/Rvb 2介导的INO 80组装途径和（ii）测试Ino 80 p和Swr 1 p插入是否以及如何插入 区域调节特定的复杂装配和活动。这将使用体外 生物物理和生物化学技术。结果将揭示INO 80和SWR 1组装中的步骤， 受Rvb 1/Rvb 2监管，并在此过程中确定装配和功能中的质量控制要点 这些复合物在体内。本研究得出的原则将广泛适用于理解 Rvb 1/Rvb 2复合体如何在其他细胞过程中发挥作用，以及缺陷的Rvb 1/Rvb 2如何发挥作用 导致特定疾病。 这项研究将在加州大学旧金山分校进行，由Geeta Narlikar博士直接监督，他是一位使用 生物化学工具来剖析染色质调节机制。Narlikar实验室和UCSF配备了所有 实现拟议的研究和培训目标所需的工具和资源。