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: 10613912
• 负责人: Gerardo Perez Goncalves
• 金额: $ 4.77万
• 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/10613912
• 关键词: Aerobic Bacteria; Allosteric Regulation; Area; Baltimore; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Catalysis; Catalytic Domain; Chemistry; Classification; Clofarabine; Collaborations; Complex; Cone; Cryoelectron Microscopy; DNA Repair; DNA biosynthesis; Data; Deoxyribonucleotides; Deuterium; Development; Drug Design; Enzyme Inhibition; Enzymes; Eukaryota; FDA approved; Future; Generations; Genomic Instability; Holoenzymes; Human; Human Activities; Hydrogen; Immunoglobulin Class Switching; Impairment; Investigation; Knowledge; Laboratories; 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; biophysical analysis; biophysical techniques; cancer therapy; chemotherapeutic agent; cofactor; 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.

项目摘要 适当维护脱氧核糖核苷酸三磷酸（DNTP）池对于高保真DNA是必需的 复制和维修。即使是DNTP池的微小变化也会导致诱变率很高，也就是说 通常在人类癌症中看到。 DNTP池的关键调节剂是核糖核苷酸还原（RNR），唯一 能够通过自由基化学来从头产生脱氧核糖核苷酸的酶。 RNR是保守的 在大多数生活形式中 催化所必需的。我们对RNR的大多数机械理解都来自IA类RNR，这是 在人类中发现的班级。人类RNR（HSRNR）的活性在ATP的结合上受到变构调节 或DATP到催化亚基（α），其中这些效果的结合分别充当开关开关。 这些作用的结合还引起了两个形态上相同的α6环的形成α6-atp和 α6-DATP。两个六聚体的稳定性各不相同：只有α6-ATP才能被自由基拆卸 产生亚基（β）形成全酶，而α6-DATP则不受添加β亚基来干扰。 化学治疗剂克洛法拉滨三磷酸是一种datp模拟，假设是变构的 抑制HSRNR，诱导α6-DATP样的“持续己酯”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有前途的方法， 但是，尚未完全确定基于活性调节的分子机制。蛋白质 还已经确定了HSRNR的调节剂，但是在任何结合方式上都没有结构数据 蛋白质调节剂和基于蛋白质的HSRNR调节的分子机制的有限表征。 因此，我们提出的研究旨在回答有关活性分子机制的问题 使用生化和生物物理技术调节HSRNR ATP/DATP以及通过蛋白质调节剂调节HSRNR活性。这项工作的结果将提供关键 详细信息对HSRNR的活性调节，以及与蛋白质复杂的RNR的第一个结构 监管机构。这项工作将在麻省理工学院生物学系的凯瑟琳·德伦南教授的实验室进行 并使用Daniel Derege博士和质谱设施的Patrick Wintrode博士提供的服务 马里兰大学：巴尔的摩药学院，并与博士博士实验室合作 国家儿童健康与人类发展研究院的玛丽·达索（Mary Dasso）。