Understanding the function of histone H3 as an oxidoreductase enzyme

项目总结 本申请建议研究新发现的组蛋白H3作为氧化还原酶的功能 酶，催化铜(铜+2)离子还原为可生物的亚铜(铜+1)形式。真核生物 组蛋白H3-H4四聚体在相对的H3蛋白与H3蛋白的界面上含有一个推测的Cu2+结合部位 未知函数。真核生物的出现与全球氧合作用同时出现，这对细胞 铜的利用，提高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，组蛋白是有效利用细胞内铜所必需的，这取决于可利用性 处于还原的+1氧化态的铜离子。通过蛋白质在细胞内运输的是铜+1离子 目的蛋白的伴侣蛋白。我们证明了H3-H4四聚体，由重组 在体外，组蛋白与Cu2+结合并催化其还原为Cu1+。基因的功能丧失和功能获得突变 推测的活性中心残基相应地改变了铜的结合和酶的活性，以及 细胞内Cu1+水平和铜依赖的活动，如线粒体呼吸和超氧化物歧化 歧化酶1(SOD1)在酿酒酵母中起作用。我们的数据揭示了组蛋白H3-H4四聚体的功能 在文献中几乎没有先例，揭示了真核基因组包裹在一种酶周围。我们现在 建议发展对组蛋白这一新功能的机械性理解，以及它是如何调节和 与细胞铜稳态有关。在目标1中，我们试图通过以下方式来了解催化的机理 铜结合的H3-H4四聚体的结构测定及其周围残基的贡献 活动站点。在目标2中，我们将了解酶的活性是如何调节的，特别是通过翻译后 组蛋白和某些组蛋白变体的修饰。组蛋白的酶活性表明 必须是一个以前未发现的生物网络，它将Cu2+穿梭到组蛋白上，然后将 反应产物(Cu1+)进入细胞的不同部分，供细胞核、细胞质和 线粒体。在目标3中，我们计划系统地鉴定与这种新型铜相关的蛋白质效应物 利用高通量CRISPR干扰(CRISPRi)技术在酵母中构建生物网络。我们的目标是 确定将组蛋白的酶活性与其他细胞功能相结合的基因和途径。 我们的提案将开始为理解染色质的结构和功能奠定科学基础 一种酶及其对真核生物生物学的影响，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，如癌症和神经退行性变，其中铜 动态平衡被改变了。

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.