Understanding the function of histone H3 as an oxidoreductase enzyme

项目摘要 本申请旨在研究新发现的组蛋白H3作为氧化还原酶的功能 酶，催化二价铜（Cu+2）离子还原成可生物降解的亚铜（Cu+1）形式。真核 组蛋白H3-H4四聚体在与H3蛋白的界面处含有推定的Cu 2+结合位点， 未知函数真核生物与全球氧合的同时出现， 铜的利用，提出了可能性，组蛋白可能在细胞铜稳态功能。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 铜离子的还原态，+1氧化态。正是Cu+1离子被蛋白质在细胞内运输 目的地靶蛋白的分子伴侣。我们发现，H3-H4四聚体，从重组组装， 组蛋白结合Cu 2+并在体外催化其还原为Cu 1+。功能丧失和获得性突变 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Cu 1+水平和铜依赖的活动，如线粒体呼吸和超氧化物 歧化酶1（SOD 1）在S.啤酒。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着一种酶。我们现在 我建议对组蛋白的这种新功能以及它是如何被调节的有一个机械的理解， 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： 确定了铜结合的H3-H4四聚体的结构以及其内部和周围残基的贡献 活动现场。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， 它必须是一个以前未被发现的生物网络，它将Cu 2+穿梭到组蛋白中，然后将 反应产物（Cu 1+）被细胞的不同部分用于细胞核、细胞质和细胞质中的蛋白质。 线粒体在目标3中，我们计划系统地鉴定这种新型铜离子所涉及的蛋白质效应物 通过利用高通量CRISPR干扰（CRISPRi）技术在酵母中构建生物网络。我们的目标 以确定整合组蛋白酶活性与其他细胞功能的基因和途径。 我们的建议将开始为理解染色质的结构和功能建立科学基础， 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，如癌症和神经变性，其中铜 体内平衡被改变了

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.