Understanding the function of histone H3 as an oxidoreductase enzyme

了解组蛋白 H3 作为氧化还原酶的功能

• 批准号: 10320937
• 负责人: Siavash Kurdistani
• 金额: $ 45.23万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320937
• 关键词: Active Sites; Archaea; Binding; Binding Sites; Biochemical; Biological; Biology; CRISPR interference; CRISPR screen; CRISPR/Cas technology; Catalysis; Catalytic Domain; Cell Nucleus; Cell Separation; Cell physiology; Cells; Chemistry; Chromatin; Chromatin Structure; Complex; Consequentialism; Copper; Coupled; Cytoplasm; DNA; Data; Destinations; Dialysis procedure; Dimensions; Disease; Electrons; Environment; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Foundations; Gene Expression; Generations; Genes; Genetic; Genetic Screening; Genome; Histone H3; Histones; Homeostasis; Human; Human Pathology; In Vitro; Instruction; Ions; Link; Literature; Malignant Neoplasms; Metabolism; Mitochondria; Mitochondrial Proteins; Modification; Molecular; Molecular Chaperones; Molecular Genetics; N-terminal; NADP; Nerve Degeneration; Oxidants; Oxidoreductase; Pathway interactions; Physiological; Post-Translational Protein Processing; Process; Proteins; Reaction; Recombinants; Reducing Agents; Regulation; Respiration; Role; Saccharomyces cerevisiae; Side; Sodium Chloride; Structure; System; Tail; Technology; Testing; Variant; Xenopus; Yeasts; base; chemical reaction; cuprous ion; deep sequencing; design; dimer; enzyme activity; epigenetic regulation; epigenome; gain of function mutation; histone modification; human disease; novel; oxidation; protein H(3); protein complex; screening; superoxide dismutase 1; yeast genetics

PROJECT SUMMARY This application proposes to investigate the newly discovered function of histone H3 as an oxidoreductase enzyme, catalyzing the reduction of cupric (Cu+2) ions to the biousable cuprous (Cu+1) form. The eukaryotic histone H3-H4 tetramer contains a putative Cu2+ binding site at the interface of the apposing H3 proteins with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We have extensive evidence that histones are required for efficient use of copper inside cells, which depend on availability of copper ions in their reduced, +1 oxidation state. It is the Cu+1 ions that are trafficked intracellularly by protein chaperones to destination target proteins. We show that the H3-H4 tetramer, assembled from recombinant histones, binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ levels and copper-dependent activities such as mitochondrial respiration and superoxide dismutase 1 (Sod1) function in S. cerevisiae. Our data have uncovered a function of the histone H3-H4 tetramer with little precedence in literature, revealing that the eukaryotic genome is wrapped around an enzyme. We now propose to develop a mechanistic understanding of this new function of histones and how it is regulated and linked to cellular copper homeostasis. In Aim 1, we seek to understand the mechanism of catalysis by determining the structure of copper-bound H3-H4 tetramer and the contributions of the residues in and around the active site. In Aim 2, we will discern how the enzyme activity is regulated, especially through post-translational modifications of histones and certain histone variants. The enzymatic activity of histones indicates that there must be a previously undiscovered biological network that shuttles Cu2+ to histones and then distributes the reaction product (Cu1+) to different parts of the cell for use by proteins in the nucleus, cytoplasm and mitochondria. In Aim 3, we plan to systematically identify the protein effectors involved in this novel copper biological network in yeast by utilizing a high-throughput CRISPR-interference (CRISPRi) technology. We aim to identify the genes and pathways that integrate the enzymatic activity of histones with other cellular functions. Our proposal will begin to build the scientific foundation for understanding chromatin structure and function as an enzyme and its impact on eukaryotic biology with instructive consequences for the evolution of the eukaryotic cell as well as a range of human pathologies such as cancer and neurodegeneration in which copper homeostasis is altered.

项目概要 本申请旨在研究组蛋白 H3 作为氧化还原酶的新发现功能 酶，催化铜 (Cu+2) 离子还原为可生物利用的亚铜 (Cu+1) 形式。真核生物 组蛋白 H3-H4 四聚体在相应的 H3 蛋白与 未知功能。真核生物与全局氧合的同时出现，对细胞提出了挑战 铜的利用，提出了组蛋白可能在细胞铜稳态中发挥作用的可能性。我们有 大量证据表明，细胞内铜的有效利用需要组蛋白，这取决于可用性 处于还原态、+1氧化态的铜离子。 Cu+1 离子通过蛋白质在细胞内运输 目标蛋白的伴侣。我们展示了由重组体组装而成的 H3-H4 四聚体 组蛋白，结合 Cu2+ 并在体外催化其还原为 Cu1+。功能丧失和获得功能突变 推定的活性位点残基相应地改变了铜结合和酶活性，以及 细胞内 Cu1+ 水平和铜依赖性活动，例如线粒体呼吸和超氧化物 歧化酶 1 (Sod1) 在酿酒酵母中发挥作用。我们的数据揭示了组蛋白 H3-H4 四聚体的功能 揭示真核基因组包裹在酶周围的文献很少有先例。我们现在 提议对组蛋白的这种新功能及其如何调控和形成机制进行理解 与细胞铜稳态有关。在目标 1 中，我们试图通过以下方式了解催化机制： 确定铜结合的 H3-H4 四聚体的结构以及内部和周围残基的贡献 活性位点。在目标 2 中，我们将了解酶活性的调节方式，特别是通过翻译后调节 组蛋白和某些组蛋白变体的修饰。组蛋白的酶活性表明 一定是一个以前未被发现的生物网络，它将 Cu2+ 运送到组蛋白，然后分配 反应产物（Cu1+）到达细胞的不同部分，供细胞核、细胞质和细胞中的蛋白质使用 线粒体。在目标 3 中，我们计划系统地鉴定参与这种新型铜的蛋白质效应子 利用高通量 CRISPR 干扰 (CRISPRi) 技术在酵母中构建生物网络。我们的目标 鉴定将组蛋白的酶活性与其他细胞功能整合的基因和途径。 我们的提案将开始为理解染色质结构和功能奠定科学基础 一种酶及其对真核生物学的影响，对真核生物的进化具有指导意义 细胞以及一系列人类病理学，例如癌症和神经变性，其中铜 体内平衡被改变。