Structural Characterization of the NuA4 Histone Acetyltransferase Complex

NuA4 组蛋白乙酰转移酶复合物的结构表征

• 批准号: 7760182
• 负责人: Johnathan Robert Chittuluru
• 金额: $ 2.84万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760182
• 关键词: Acetylation; Acetyltransferase; Address; Affinity; Antibodies; Binding; Biochemical; Biochemistry; Catalysis; Cell Survival; Cell physiology; Cells; Chromatin; Chromatin Structure; Complex; Cryoelectron Microscopy; DNA; DNA Structure; Data; Docking; Electron Microscopy; Engineering; Enzymes; Eukaryota; Future; Histone Acetylation; Histone H4; Histones; Human; In Vitro; Knowledge; Lead; Link; Malignant Neoplasms; Mediating; Methods; Nucleosome Core Particle; Nucleosomes; Process; Regulation; Research; Research Design; Resolution; Saccharomyces cerevisiae; Series; Structure; Yeast Model System; Yeasts; base; cancer therapy; cancer type; design; histone acetyltransferase; histone modification; improved; mutant; particle; polypeptide; public health relevance; research study; three dimensional structure; three-dimensional modeling

DESCRIPTION (provided by applicant): The dynamic structure of DNA in eukaryotes is integral to DNA transactions and therefore fundamentally linked to many types of cancer. Chromatin modifying enzymes, which catalyze an array of covalent posttranslational histone modifications, are responsible for mediating many of the structural transitions between different levels of chromatin packaging. Histone acetylation is the most extensively studied histone modification, and the only histone acetyltransferase (Esa1) required for cell viability in the yeast Saccharomyces cerevisiae resides within the nucleosome acetyltransferase of histone H4 (NuA4) complex. NuA4 is a large (1.3 MDa) multisubunit complex whose biochemistry has been the subject of extensive research, but little is know about its structure. This proposal seeks to utilize single-particle electron microscopy (EM) to address the following specific aims concerning NuA4 and its catalytic subcomplex, Piccolo: (1) develop an intermediate resolution (-10 A) cryo-EM model of the yeast NuA4 complex and study the interaction between NuA4 and its substrate, the nucleosome, (2) investigate the subunit organization of the NuA4 HAT complex and explore how this organization facilitates NuA4 function, and (3) determine the three-dimensional structure of the catalytic Piccolo subcomplex alone and bound to the nucleosome. To briefly describe the research design and methods for addressing these aims, EM will be used to characterize the NuA4, Piccolo, and Piccolo-NCP complexes to determine their three dimensional structures. Also, in a series of separate experiments for localizing NuA4 subunits, EM will characterize NuA4 subunit deletion mutants and NuA4 bound by antibodies specific for an engineered affinity tags present on targeted subunits. Additionally, EM will characterize the interaction between NuA4 and the nucleosome. Combined, these efforts will thoroughly characterize NuA4's structure and provide a framework for interpreting previous, ongoing, and future biochemical studies of NuA4 and related complexes. PUBLIC HEALTH RELEVANCE: This study will investigate how chromatin structure is regulated. No process is more vital to the cell than the regulation of chromatin structure, and like many essential cellular processes, its disruption can lead to cancer. Studies (like this one) of fundamentally important cellular processes related to cancer contribute greatly to the body of fundamental knowledge required to design effective cancer therapies.

描述（由申请人提供）：真核生物中DNA的动态结构与DNA交易不可或缺，因此从根本上与许多类型的癌症联系在一起。染色质修饰酶催化一系列共价翻译后组蛋白修饰，负责介导不同水平的染色质包装之间的许多结构过渡。组蛋白乙酰化是最广泛研究的组蛋白修饰，并且在酿酒酵母中细胞活力所需的唯一组蛋白乙酰基转移酶（ESA1）位于组蛋白H4（NUA4）的核小体乙酰基转移酶内。 NUA4是一个大型（1.3 MDA）多生育综合体，其生物化学一直是广泛研究的主题，但对其结构知之甚少。 This proposal seeks to utilize single-particle electron microscopy (EM) to address the following specific aims concerning NuA4 and its catalytic subcomplex, Piccolo: (1) develop an intermediate resolution (-10 A) cryo-EM model of the yeast NuA4 complex and study the interaction between NuA4 and its substrate, the nucleosome, (2) investigate the subunit organization of the NuA4 HAT complex并探讨该组织如何促进NUA4功能，（3）单独确定催化短笛亚型子相结合并与核小体结合的三维结构。为了简要描述解决这些目标的研究设计和方法，将使用EM来表征NUA4，Piccolo和Piccolo-NCP复合物，以确定其三维结构。同样，在一系列用于定位NUA4亚基的单独实验中，EM将表征NUA4亚基缺失突变体和NUA4，而NUA4受到针对目标亚基上存在的工程亲和力标签的抗体结合。另外，EM将表征NUA4与核小体之间的相互作用。这些努力结合在一起，将彻底描述NUA4的结构，并为解释NUA4及相关复合物的以前，正在进行和未来的生化研究提供了一个框架。公共卫生相关性：这项研究将研究如何调节染色质结构。没有比染色质结构调节的过程对细胞更重要，并且像许多必需的细胞过程一样，其破坏会导致癌症。与癌症有关的根本重要细胞过程（如这样）的研究对设计有效的癌症疗法所需的基本知识造成了很大的贡献。