Mechanism of action of a major folate enzyme

主要叶酸酶的作用机制

• 批准号: 8013378
• 负责人: SERGEY A KRUPENKO
• 金额: $ 9.65万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-08 至 2011-02-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013378
• 关键词: Address; Anabolism; Antibodies; Apoptosis; Bacteria; Biogenesis; Biological; Biological Assay; Cancerous; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell physiology; Cells; Chromosomes; Congenital Abnormality; Cytosol; Data; Disease; Enzymes; Folate; Folate Biosynthesis Pathway; Formates; Formyltetrahydrofolates; Genes; Goals; Grant; Human; Hydrolase; Liver; Malignant Neoplasms; Mammalian Cell; Measurement; Megaloblastic Anemia; Metabolic; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular Profiling; Neural tube; Nucleotides; Oxidoreductase; Prevention; Production; Protein Biosynthesis; Protein Biosynthesis Pathway; Proteins; Purine Antagonist; Purines; Reaction; Regulation; Research Personnel; Role; Small Interfering RNA; Supplementation; Techniques; Testing; Tetrahydrofolates; Tissues; Transfer RNA; Translation Initiation; Tumor Suppressor Proteins; Vascular Diseases; base; cancer cell; enzyme activity; folic acid metabolism; human tissue; in vivo; inhibitor/antagonist; mutant; programs; protein expression; purine; tumor

DESCRIPTION (provided by applicant): The broad objectives of this proposal are to understand the metabolic role of one of the most abundant folate enzymes, FDH. FDH converts 10-formyltetrahydrofolate (10-fTHF) to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction. Our recent studies have demonstrated that FDH possesses tumor suppressor-like activity: it is strongly and ubiquitously down regulated in tumors and induces apoptosis in FDH-deficient cancer cells. Therefore, we proposed that the enzyme is one of the intrinsic mechanisms that protect against excessive and uncontrolled cellular proliferation. Since the FDH substrate, 10-fTHF, formylates methionyl-tRNA, presumably a required step in initiation of translation in mitochondria, we propose that FDH regulates protein biosynthesis in mitochondria through the control of intracellular 10-fTHF levels. We further hypothesize that the product of the FLJ38508 gene (locus 12q23.3) is a mitochondria! FDH, and that the cytosolic and mitochondria! enzymes regulate distribution of 10-fTHF between cytosolic and mitochondrial compartments directing 10-fTHF to the de novo purine biosynthesis or mitochondrial protein biosynthesis pathway. We also propose that FDH hydrolase reaction occurs in vivo in mitochondria and that the biological role of this reaction is to supply formate for biosynthesis of 10-fTHF in cytosol. The following specific aims are proposed to test the hypotheses. (1) Manipulate the levels of 10-fTHF, through FDH expression, to establish its importance in control of protein biosynthesis in mitochondria. (2) Explore the role of the mitochondrial FDH in cellular function. (3) Investigate whether FDH- catalyzed 10-fTHF hydrolase reaction occurs in vivo. Cultured mammalian cells with different supplementation of folate and purines will be used as a model in this proposal. FDH expression in mammalian cells, measurement of folate and nucleotide pools, analysis of folate enzymes, assays of ATP production and protein expression in mitochondria, assays of apoptosis and mitochondrial integrity, enzyme activity assays, immunochemical methods, siRNA techniques will be used to achieve the goals of the project. The well-known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer, as well as crucial role of mitochondria in regulation of apoptosis, and growing body of evidence for mitochondrial basis of many diseases make these studies particularly relevant.

描述（由申请人提供）：本提案的主要目的是了解最丰富的叶酸酶之一FDH的代谢作用。FDH在NADP依赖性脱氢酶反应或NADP非依赖性水解酶反应中将10-甲酰四氢叶酸（10-fTHF）转化为四氢叶酸。我们最近的研究表明，FDH具有肿瘤抑制剂样活性：它在肿瘤中强烈且普遍下调，并诱导FDH缺陷癌细胞的凋亡。因此，我们认为这种酶是防止过度和不受控制的细胞增殖的内在机制之一。由于FDH底物10-fTHF甲酰化甲硫氨酰-tRNA，推测这是线粒体翻译起始所需的步骤，因此我们提出FDH通过控制细胞内10-fTHF水平来调节线粒体中的蛋白质生物合成。我们进一步假设FLJ 38508基因（位点12q23.3）的产物是线粒体！FDH，以及胞质和线粒体！酶调节10-fTHF在胞质和线粒体区室之间的分布，将10-fTHF引导至从头嘌呤生物合成或线粒体蛋白质生物合成途径。我们还提出，FDH水解酶反应发生在体内的线粒体和该反应的生物学作用是提供10-fTHF在胞质溶胶中的生物合成的甲酸。为检验这些假设，提出了以下具体目标。(1)通过FDH表达操纵10-fTHF的水平，以确定其在线粒体中蛋白质生物合成控制中的重要性。(2)探索线粒体FDH在细胞功能中的作用。(3)调查FDH催化的10-fTHF水解酶反应是否在体内发生。本研究以添加不同叶酸和嘌呤的哺乳动物细胞为模型。FDH在哺乳动物细胞中的表达、叶酸和核苷酸库的测量、叶酸酶的分析、线粒体中ATP产生和蛋白表达的测定、细胞凋亡和线粒体完整性的测定、酶活性测定、免疫化学方法、siRNA技术将用于实现该项目的目标。众所周知，叶酸在预防巨幼细胞性贫血、血管疾病、神经管出生缺陷和癌症中的作用，以及线粒体在调节细胞凋亡中的关键作用，以及越来越多的证据表明许多疾病的线粒体基础使这些研究特别相关。