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）：维持适当和平衡的脱氧核糖核苷酸（DNTP）池对于忠实的DNA复制和修复至关重要。 DNTP池的正常控制丧失会导致细胞死亡，基因组不稳定性和人类癌症的易感性。核糖核苷酸还原酶（RNR）的调节主要负责控制细胞DNTP池的相对比率和量。 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目标。 RNR酶由两个亚基组成：R1亚基结合了四个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亚基载有R1中核苷酸减少的必需酪液自由基所需的必需酪液自由基。 RNR的酶活性可以通过变构，转录，蛋白质抑制剂关联和亚基的亚细胞隔室调节。酿酒酵母的萌芽酵母已成为一种原型模型系统，以研究调节RNR活性的复杂机制。该建议着重于通过使用生化，细胞生物学和遗传方法的组合来控制RNR活性的机制以及因此细胞DNTP池。我们的中心假设是，这些调节机制已整合以在不同的生长条件下维持最佳的DNTP池，以确保高保真性DNA的合成和修复。提出了三个具体目的：（1）检验以下假设：SML1蛋白通过阻碍R1活性位点的再生并定义R1-SML1相互作用和SML1降解的分子决定因素来抑制RNR酶； （2）检查通过细胞周期和DNA损伤检查点的R2亚基的亚细胞定位调节； （3）表征新鉴定的小蛋白SLD1在RNR调节中的作用。 SLD1属于一个小蛋白RNR调节剂，包括S. cerevisiae Sml1和Sld1，S。PombeSPD1及其同源物，由其他真菌基因组编码。重点是对这些进化保守的小型RNR调节蛋白的控制如何控制RNR活性的机械理解。从对酵母RNR的研究中学到的经验教训将是理解真核细胞中RNR调节和DNTP池控制的范例，还可能提出新的RNR抑制作用方法，抗肿瘤和抗病毒药物。公共卫生相关性：核糖核苷酸还原酶是一种必不可少的酶，为所有生物体提供DNA的基础。该酶也是人类癌症临床治疗的靶向靶标。该项目的总体目标是了解该酶在细胞内部如何控制该酶的活性，并找到针对该酶的药物开发的新策略。