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

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 到蛋白质翻译。这些蛋白质机器的组装需要能够协调 亚基之间的必要相互作用，同时防止不适当的相互作用。细胞设法做到的一种方式是 这是通过利用伴侣，一组不同的蛋白质，帮助非共价折叠和解折叠， 以及大分子结构的组装和拆解。Rvb1和Rvb2是两种必需的AAA+ATPase 从酿酒酵母(S.cerevisiae)中分离出来的(Rvb1/Rvb2)被认为是不同多基因的组装伴侣。 亚基复合体，包括部分染色质重构体。Rvb1和Rvb2还与各种 细胞过程，包括转录、DNA损伤和细胞信号传递，及其缺陷是强烈的 与多种癌症有关，包括肝细胞癌和急性髓系白血病。然而，这是如何 Rvb1/Rvb2复合体作为伴侣的功能还知之甚少。本提案将探讨Rvb1/Rvb2 在INO80和SWR1染色质重构的组装和活性中的作用 复合体。INO80和SWR1是由多个亚基组成的染色质重构体，包括Rvb1/Rvb2 六角体，它利用三磷酸腺苷水解产生的能量来重塑核小体底物。有证据表明 在INO80复杂组装背景下的Rvb1/Rvb2伴侣样活性。一种建议的INO80中间体 组装体含有Rvb1/Rvb2，表现出增强的Rvb1/Rvb2 ATPase活性。然而，Rvb1/Rvb2的角色是S 整个复杂的组装过程仍不清楚。INO80和SWR1具有相似的整体架构，但 独特的亚基和核小体重塑活动。Rvb1/Rvb2和Rvb1/Rvb2之间的相互作用 核心亚单位Ino80p和Swr1p的插入区域可能调节独特的复杂组装和活性 INO80和SWR1。这项提案将(I)确定和描述 Rvb1/Rvb2介导的INO80组装途径和(Ii)检测Ino80p和Swr1p是否以及如何插入 区域调节特定的复杂的组装和活动。这将使用体外试验的组合来完成 生物物理和生化技术。结果将揭示INO80和SWR1组装中的步骤 受Rvb1/Rvb2规范，并在此过程中确定装配和功能中的质量控制要点 体内的这些复合体。从这项研究得出的原则将广泛适用于理解 Rvb1/Rvb2复合体如何在其他细胞过程中发挥作用以及有缺陷的Rvb1/Rvb2如何发挥作用 会导致特定的疾病。 这项研究将在加州大学旧金山分校在吉塔·纳利卡尔博士的直接监督下进行，他是一名使用 分析染色质调节机制的生化工具。纳里卡实验室和加州大学旧金山分校配备了所有 完成拟议的研究和培训目标所需的工具和资源。

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.