In Vivo Regulation and Inhibition of Ribonucleotide Reductase

核糖核苷酸还原酶的体内调节和抑制

• 批准号: 7894601
• 负责人: MINGXIA HUANG
• 金额: $ 31.76万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894601
• 关键词: Active Sites; Anabolism; Antiviral Agents; Binding; Binding Sites; Biochemical; Biological Models; Catalytic Domain; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cells; Cellular biology; Clinical Treatment; Complex; DNA; DNA Damage; DNA biosynthesis; DNA damage checkpoint; Deoxyribonucleotides; Development; Ensure; Enzymes; Equilibrium; Eubacterium; Eukaryota; Eukaryotic Cell; Family; Fission Yeast; Fungal Genome; Genetic; Genetic Transcription; Genomic Instability; Goals; Growth; Homologous Gene; Housing; Human; Hypersensitivity; In Vitro; Lead; Learning; Maintenance; Malignant Neoplasms; Molecular; Molecular Genetics; Natural regeneration; Normal Cell; Nucleotides; Organism; Pattern; Phosphorylation; Predisposition; Proteins; Public Health; Regulation; Relative (related person); Ribonucleotide Reductase; Ribonucleotide Reductase Subunit; Role; Saccharomyces cerevisiae; Saccharomycetales; Sequence Homology; Substrate Specificity; Testing; Yeasts; base; cofactor; drug development; enzyme activity; genetic regulatory protein; in vivo; inhibitor/antagonist; novel strategies; repaired; research study; response

DESCRIPTION (provided by applicant): The maintenance of adequate and balanced deoxyribonucleotide (dNTP) pools is essential for faithful DNA replication and repair. Loss of normal control of the dNTP pools can lead to cell death, genomic instability, and predisposition to cancer in humans. Regulation of ribonucleotide reductase (RNR) is largely responsible for controlling the relative ratios and amounts of the cellular dNTP pools. The central role of RNR in dNTP biosynthesis has also made it a successful target in the treatment of a number of malignancies. The RNR enzyme comprises of two subunits: the R1 subunit binds the four NDP substrates as well as the allosteric effectors (NTPs and dATP) that govern substrate specificity and turnover rate, and the R2 subunit houses the essential tyrosyl radical required to initiate nucleotide reduction in R1. The enzymatic activity of RNR can be modulated by allostery, transcription, protein inhibitor association, and subcellular compartmentation of its subunits. The budding yeast S. cerevisiae has emerged as a prototypical model system with which to investigate the complex mechanisms regulating the RNR activity. This proposal focuses on the mechanisms that control the RNR activity and consequently cellular dNTP pools by using a combination of biochemical, cell biology, and genetic approaches. Our central hypothesis is that these regulatory mechanisms are integrated to maintain optimal dNTP pools under different growth conditions so as to ensure high fidelity DNA synthesis and repair. Three specific aims are proposed: (1) To test the hypothesis that the Sml1 protein inhibits the RNR enzyme by impeding regeneration of the R1 active site and to define molecular determinants of the R1-Sml1 interaction and Sml1 degradation; (2) To examine the regulation of subcellular localization of the R2 subunit by the cell cycle and DNA damage checkpoints; (3) To characterize the role of the newly identified small protein Sld1 in RNR regulation. Sld1 belongs to a family of small protein RNR regulators, including the S. cerevisiae Sml1 and Sld1, the S. pombe Spd1, and their homologs encoded by other fungal genomes. The emphasis is to gain a mechanistic understanding of how the RNR activity is controlled by these evolutionarily conserved small size RNR regulatory proteins. Lessons learned from studies of the yeast RNR will serve as a paradigm for understanding of RNR regulation and dNTP pool control in eukaryotic cells, and may also suggest new approaches for RNR inhibition and for antitumor and antiviral drug development. PUBLIC HEALTH RELEVENCE: Ribonucleotide reductase is an essential enzyme that provides the building blocks for DNA in all organisms. This enzyme is also a proven target of clinical treatment of human cancers. The overall goal of this project is to understand how the activity of this enzyme is controlled inside the cell and finding new strategy for drug development targeting this enzyme.

描述（由申请人提供）：维持充足且平衡的脱氧核糖核苷酸（dNTP）库对于忠实的 DNA 复制和修复至关重要。 dNTP 池失去正常控制可能导致细胞死亡、基因组不稳定以及人类患癌症的倾向。核糖核苷酸还原酶 (RNR) 的调节主要负责控制细胞 dNTP 池的相对比例和数量。 RNR 在 dNTP 生物合成中的核心作用也使其成为治疗多种恶性肿瘤的成功靶点。 RNR 酶由两个亚基组成：R1 亚基结合四种 NDP 底物以及控制底物特异性和周转率的变构效应子（NTP 和 dATP），R2 亚基包含启动核苷酸还原所需的必需酪氨酰自由基。 R1。 RNR 的酶活性可通过变构、转录、蛋白质抑制剂关联及其亚基的亚细胞区室来调节。芽殖酵母酿酒酵母已成为研究调节 RNR 活性的复杂机制的典型模型系统。该提案重点关注通过结合使用生化、细胞生物学和遗传方法来控制 RNR 活性以及细胞 dNTP 池的机制。我们的中心假设是，这些调控机制被整合起来，以在不同的生长条件下维持最佳的 dNTP 库，从而确保高保真度的 DNA 合成和修复。提出了三个具体目标：（1）检验Sml1蛋白通过阻碍R1活性位点再生来抑制RNR酶的假设，并确定R1-Sml1相互作用和Sml1降解的分子决定因素； (2) 研究细胞周期和DNA损伤检查点对R2亚基亚细胞定位的调控； (3) 表征新发现的小蛋白Sld1在RNR调节中的作用。 Sld1 属于小蛋白 RNR 调节因子家族，包括酿酒酵母 Sml1 和 Sld1、粟酒裂殖酵母 Spd1 及其由其他真菌基因组编码的同源物。重点是从机制上理解这些进化上保守的小尺寸 RNR 调节蛋白如何控制 RNR 活性。从酵母 RNR 研究中获得的经验教训将作为理解真核细胞中 RNR 调节和 dNTP 库控制的范例，并且还可能为 RNR 抑制以及抗肿瘤和抗病毒药物开发提供新方法。公共卫生相关性：核糖核苷酸还原酶是一种重要的酶，为所有生物体中的 DNA 提供构建模块。这种酶也是人类癌症临床治疗的经过验证的靶标。该项目的总体目标是了解细胞内如何控制这种酶的活性，并找到针对这种酶的药物开发新策略。