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.

项目摘要 该应用建议研究组蛋白H3作为氧化还原酶的新发现的功能 酶，催化铜（Cu+2）离子的缩小为二铜（Cu+1）形式。真核生物 组蛋白H3-H4四聚体在与H3蛋白的界面上包含一个假定的Cu2+结合位点 未知功能。真核生物与全球氧合的重合出现，这挑战了细胞 铜的利用，增加了组蛋白在细胞铜稳态中起作用的可能性。我们有 广泛的证据表明，需要有效使用铜内部细胞内部的组蛋白，这取决于可用性 铜离子在其降低的+1氧化状态中的含量。是Cu+1个离子被蛋白质细胞内运输 伴侣到目标靶蛋白。我们表明，由重组组装的H3-H4四聚体 组蛋白结合Cu2+并催化其还原为Cu1+体外。损失和功能收益突变 推定的活动位点残基相应改变了铜结合和酶活性，以及 细胞内Cu1+水平和铜依赖性活性，例如线粒体呼吸和超氧化物 酿酒酵母中的歧化酶1（SOD1）功能。我们的数据发现了组蛋白H3-H4四聚体的功能 在文献中几乎没有优先，表明真核基因组被包裹在酶周围。我们现在 提议对这种新功能的组蛋白及其调节以及 与细胞铜稳态有关。在AIM 1中，我们试图通过 确定铜结合的H3-H4四聚体的结构以及残留物的贡献 活跃的站点。在AIM 2中，我们将辨别如何调节酶活性，尤其是通过翻译后 组蛋白和某些组蛋白变体的修饰。组蛋白的酶活性表明那里 必须是先前未被发现的生物网络，将Cu2+穿梭到组蛋白，然后分配 反应产物（Cu1+）与细胞的不同部分，以供蛋白质在细胞核，细胞质和 线粒体。在AIM 3中，我们计划系统地识别这种新型铜所涉及的蛋白质效应子 利用高通量CRISPR介入（CRISPRI）技术，酵母中的生物网络。我们的目标 确定将组蛋白与其他细胞功能相结合的基因和途径。 我们的建议将开始建立科学基础，以理解染色质结构和功能 一种酶及其对真核生物学的影响，对真核的进化产生了有益的后果 细胞以及一系列人类病理，例如癌症和神经退行性，其中铜 稳态发生了变化。