Mechanism of action of a major folate enzyme

描述(由申请人提供)：本提案的广泛目标是了解最丰富的叶酸酶之一--FDH的代谢作用。FDH在NADP依赖的脱氢酶反应或NADP非依赖的水解酶反应中将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具有肿瘤抑制物样活性：它在肿瘤中强烈且普遍下调，并诱导FDH缺陷的癌细胞凋亡。因此，我们认为该酶是防止细胞过度和失控增殖的内在机制之一。由于FDH底物10-fTHF甲酰化甲硫酰-tRNA，可能是启动线粒体翻译的必要步骤，我们认为FDH通过控制细胞内10-fTHF水平来调节线粒体中蛋白质的生物合成。我们进一步假设，FLJ38508基因(12q23.3位点)的产物是线粒体！外佣，还有那个胞浆和线粒体！酶调节10-fTHF在胞浆和线粒体之间的分布，将10-fTHF导向从头合成嘌呤或线粒体蛋白质的生物合成途径。我们还提出，FDH水解酶反应在体内发生在线粒体中，该反应的生物学作用是为胞浆中10-fTHF的生物合成提供甲酸盐。为了检验这些假设，本文提出了以下具体目标。(1)通过FDH的表达来调控10-fTHF的水平，以确定其在控制线粒体蛋白质生物合成中的重要性。(2)探讨线粒体脱氢酶在细胞功能中的作用。(3)研究FDH催化的10-fTHF水解酶在体内是否发生反应。在这项建议中，将使用含有不同叶酸和嘌呤补充的培养的哺乳动物细胞作为模型。在哺乳动物细胞中的FDH表达，叶酸和核苷酸池的测量，叶酸酶的分析，线粒体中ATP的产生和蛋白表达的分析，细胞凋亡和线粒体完整性的分析，酶活性的分析，免疫化学方法，siRNA技术将被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线粒体是许多疾病的基础，使得这些研究特别相关。

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.