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.

项目总结 适当地维护脱氧核糖核酸三磷酸(DNTP)池是高保真DNA所必需的 复制和修复。即使dNTP池中的微小变化也会导致高突变率，这是 在人类癌症中很常见。DNTP池的一个关键调节因子是核苷酸还原酶(RNR)，它是dNTP池中唯一的 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 在大多数生命形式中，根据产生自由基的辅因子被分为三类 对催化来说是必要的。我们对RNR的大部分机械理解来自于Ia类RNR，这是 在人类身上发现的这一类。人RNR(HsRNR)活性受ATP结合的变构调节 或dATP与催化亚单位(α)结合，其中这些效应器的结合分别起到打开或关闭开关的作用。 这些效应物的结合也诱导了两个形态相同的α6环的形成，α6-ATP6和 α6-dATP.这两个六聚体的稳定性不同：只有α6-ATP能被自由基分解- 生成亚单位(β)形成全酶，而α6-dATP不受β亚基的干扰。 化疗药物氯法拉滨三磷酸是一种dATP类似物，假设为变构。 抑制HsRNR，诱导形成α6-dATP样“持续性六聚体”。这些结果表明， 靶向HsRNR的变构活性部位是开发抗癌新药的一条很有前途的途径。 但是支持活性调节的分子机制还没有完全建立起来。蛋白 HsRNR的调节者也已经确定，但没有关于任何结合模式的结构数据。 蛋白质调控和基于蛋白质的HsRNR调控分子机制的有限表征。 因此，我们建议进行旨在回答有关活性分子机制的问题的研究。 HsRNR的调节，利用生化和生物物理技术探讨HsRNR的活性调节 ATP/dATP和HsRNR活性通过蛋白质调节来调节。这项工作的成果将提供关键 HsRNR活性调节的细节，以及RNR与蛋白质的复合体的第一结构 调整器。这项工作将在麻省理工学院生物系凯瑟琳·L·德里南教授的实验室进行 并使用质谱学设施的丹尼尔·德里克博士和帕特里克·温特德博士提供的服务 在马里兰大学：巴尔的摩药学院，并与博士的实验室合作。 玛丽·达索在美国国家儿童健康和人类发展研究所工作。