Biochemical and Biophysical Studies of Human Ribonucleotide Reductase

人核糖核苷酸还原酶的生化和生物物理研究

• 批准号: 10463910
• 负责人: Gerardo Perez Goncalves
• 金额: $ 4.68万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463910
• 关键词: Aerobic Bacteria; Allosteric Regulation; Area; Baltimore; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Catalysis; Catalytic Domain; Chemistry; Clofarabine; Collaborations; Complex; Cone; Cryoelectron Microscopy; Crystallization; DNA Repair; DNA biosynthesis; Data; Deoxyribonucleotides; Deuterium; Development; Drug Design; Electron Microscopy; Enzyme Inhibition; Enzymes; Eukaryota; Eye; FDA approved; Future; Generations; Genomic Instability; Holoenzymes; Human; Human Activities; Hydrogen; Immunoglobulin Class Switching; Impairment; Investigation; Knowledge; Laboratories; Lead; Life; Maintenance; Malignant Neoplasms; Maryland; Mass Spectrum Analysis; Mediating; Molecular; Morphology; Movement; Mutagenesis; National Institute of Child Health and Human Development; Negative Staining; Nucleotides; Pharmacy Schools; Proteins; Reaction; Regulation; Resolution; Ribonucleotide Reductase; Ribonucleotides; Roentgen Rays; Rotation; Services; Site; Structure; Surface; Techniques; Testing; Universities; Variant; Work; analytical ultracentrifugation; base; biophysical analysis; biophysical techniques; cancer therapy; chemotherapeutic agent; cofactor; cryogenics; enzyme activity; experimental study; improved; insight; novel; novel anticancer drug; sedimentation velocity; targeted cancer therapy; trait; tripolyphosphate

PROJECT SUMMARY Proper maintenance of deoxyribonucleotide triphosphate (dNTP) pools is necessary for high-fidelity DNA replication and repair. Even small changes in the dNTP pools can lead to high rates of mutagenesis, which is commonly seen in human cancers. A key regulator of dNTP pools is ribonucleotide reductase (RNR), the sole enzyme capable of de novo generation of deoxyribonucleotides via radical chemistry. RNRs are conserved across most forms of life, and are split up into three classes based on the cofactor that generates the radical necessary for catalysis. Most of our mechanistic understanding of RNRs comes from class Ia RNRs, which is the class found in humans. The activity of human RNR (HsRNR) is allosterically regulated by the binding of ATP or dATP to the catalytic subunit (α), where the binding of these effectors acts as an on or off switch, respectively. The binding of these effectors also induces the formation of two morphologically identical α6 rings, α6-ATP and α6-dATP. The two hexamers vary in their stability: where only α6-ATP can be disassembled by the radical- generating subunit (β) to form the holoenzyme, whereas α6-dATP is undisturbed by addition of the β subunit. The chemotherapeutic agent clofarabine triphosphate is a dATP-mimic that is hypothesized to allosterically inhibit HsRNR, inducing the formation of α6-dATP-like “persistent hexamers.” These results suggest that targeting allosteric activity sites of HsRNR is a promising approach for development of new anticancer drugs, but the molecular mechanisms underpinning activity regulation have not been fully established. Protein regulators of HsRNR have also been identified, but there is no structural data on the mode of binding of any protein regulator and limited characterization of the molecular mechanism of protein-based regulation of HsRNR. Therefore, we propose studies that aim to answer questions about the molecular mechanisms of activity regulation of HsRNR, using biochemical and biophysical techniques to probe both HsRNR activity regulation via ATP/dATP and also HsRNR activity regulation via protein regulators. The results of this work will provide key details into the activity regulation of HsRNR, along with the first structure of RNR in complex with a protein regulator. This work will be carried out in the lab of Prof. Catherine L. Drennan at the MIT Department of Biology and using the services provided by Dr. Daniel Derege and Dr. Patrick Wintrode of the Mass Spectrometry facility at the University of Maryland: Baltimore’s School of Pharmacy and in collaboration with the laboratory of Dr. Mary Dasso at the National Institutes of Child Health and Human Development.

项目概要 正确维护脱氧核糖核苷酸三磷酸 (dNTP) 库对于高保真 DNA 是必要的 复制和修复。即使 dNTP 池中的微小变化也可能导致高诱变率，即 常见于人类癌症。 dNTP 池的一个关键调节因子是核糖核苷酸还原酶 (RNR)，它是唯一的调节因子。 能够通过自由基化学从头生成脱氧核糖核苷酸的酶。 RNR 是保守的 跨越大多数生命形式，并根据产生自由基的辅因子分为三类 催化所必需的。我们对 RNR 的大部分机制理解来自 Ia 类 RNR，即 在人类中发现的类别。人类 RNR (HsRNR) 的活性通过 ATP 的结合进行变构调节 或 dATP 至催化亚基 (α)，其中这些效应子的结合分别充当开关。 这些效应子的结合还诱导形成两个形态相同的 α6 环，α6-ATP 和 α6-dATP。这两个六聚体的稳定性有所不同：其中只有 α6-ATP 可以被自由基分解。 生成亚基 (β) 以形成全酶，而 α6-dATP 不受添加 β 亚基的干扰。 化疗药物氯法拉滨三磷酸盐是一种 dATP 模拟物，假设具有变构作用 抑制 HsRNR，诱导 α6-dATP 样“持久六聚体”的形成。这些结果表明 靶向 HsRNR 的变构活性位点是开发新抗癌药物的一种有前途的方法， 但支撑活性调节的分子机制尚未完全建立。蛋白质 HsRNR 的调节因子也已被确定，但没有关于任何结合模式的结构数据 蛋白质调节剂和基于蛋白质的 HsRNR 调节分子机制的有限表征。 因此，我们提出旨在回答有关活性分子机制问题的研究 HsRNR 的调节，使用生物化学和生物物理技术来探测 HsRNR 活性调节 ATP/dATP 以及 HsRNR 活性通过蛋白质调节剂进行调节。这项工作的结果将提供关键 详细了解 HsRNR 的活性调节，以及 RNR 与蛋白质复合物的第一个结构 调节器。这项工作将在麻省理工学院生物系 Catherine L. Drennan 教授的实验室中进行 并使用质谱设施的 Daniel Derege 博士和 Patrick Wintrode 博士提供的服务 在马里兰大学巴尔的摩药学院与博士实验室合作。 美国国立儿童健康与人类发展研究所的玛丽·达索。