Mechanism of Action of a Major Folate Enzyme

主要叶酸酶的作用机制

• 批准号: 8496757
• 负责人: SERGEY A KRUPENKO
• 金额: $ 43.97万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8496757
• 关键词: 4' -phosphopantetheine; Acetaldehyde; Acetaminophen; Address; Alcohol consumption; Amino Acids; Anabolism; Animal Model; Astrocytes; Binding; Carbon; Carbon Dioxide; Catalysis; Catalytic Domain; Cell Culture Techniques; Cell Proliferation; Cell division; Cells; Coenzymes; Communities; Congenital Abnormality; CpG Islands; DNA Methylation; Diet; Disease; Enzyme Inhibition; Enzymes; Equation; Ethanol; Evaluation; Excision; Faculty; Family; Folate; Folic Acid Deficiency; Formates; Formyltetrahydrofolates; Foundations; Genes; Genomics; Grant; Health; Hepatocyte; Hepatotoxicity; Holoenzymes; Homologous Gene; Human; Injury to Liver; Kidney; Knockout Mice; Lead; Length; Liver; Malignant Neoplasms; Mammals; Megaloblastic Anemia; Metabolic; Metabolism; Methionine; Methylation; Mind; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Mus; Neural Tube Defects; Neural tube; Neurosciences; Niacinamide; Nutrient; Organism; Oxidoreductase; Paper; Pathogenesis; Pathway interactions; Physiological; Post-Translational Protein Processing; Prevention; Promoter Regions; Prosthesis; Proteins; Purines; Rattus; Reaction; Reporting; Role; Serine; Solid; Structure; Supplementation; Targeted Research; Tetrahydrofolates; Time; Tissues; Toxic effect; Tumor Suppressor Proteins; Vascular Diseases; acetaminophen overdose; adduct; aldehyde dehydrogenases; arm; base; cancer cell; cell motility; covalent bond; drug mechanism; fatty acid biosynthesis; flexibility; folic acid metabolism; follow-up; in vivo; insight; killings; liver injury; member; mutant; novel; oxidation; prevent; purine; research study; 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, 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), and how the inhibition of its catalysis is involved in pathogenesis. Our previous studies have demonstrated that the enzyme is present in many tissues but is most abundant in liver and kidney comprising in the former up to 1.2% of the total pool of cytosolic protein. At the same time it is strongly and ubiquitously down regulate in human cancers, through a CpG island methylation in its promoter region. Moreover, the inhibition of the enzyme associated with acetaminophen overdose or alcohol consumption is believed to contribute to liver toxicity. FDH converts 10- formyltetrahydrofolate (10-fTHF) to tetrahydrofolate (THF) and CO2. Importantly, this reaction removes carbon groups from the folate pool, in the form of CO2, thus decreasing folate-dependent biosynthetic capacity. Based on this, our first hypothesis is that FDH regulates overall flow of one-carbon groups through the folate pool and is one of the intrinsic mechanisms controlling cellular proliferation. The FDH reaction is also the rate-limiting step in formate clearance. Of note, formate is a toxic metabolit normally produced in humans by several physiological pathways. Thus, our second hypothesis is that inhibition of FDH in non-proliferating hepatocytes will lead to formate toxicity. We have recently found that FDH requires the prosthetic group, 4'- phosphopantetheine (4-PP), for catalysis. This flexible long arm is covalently attached to the enzyme, transferring the reaction intermediate between two catalytic sites, and its removal/modification completely "kills" enzymatic activity. Accordingly, our third hypothesis is that active metabolites of acetaminophen and ethanol inhibit FDH activity by forming adducts with its prosthetic group and this inhibition contributes to liver injury. Importantly, in the previous grant cycle we have discovered a novel enzyme, mitochondrial FDH (ALDH1L2). In the present proposal we will not only pursue FDH as a research target but will also explore the significance of the entire 10-fTHF to THF pathway by adding to the equation the mitochondrial branch of the pathway. Specific aims to address our hypothesis are: (1) Solve the crystal structure of full-length apo-FDH and the 4- PP-modified holoenzyme. (2) Determine whether FDH is inhibited in vivo by covalent adduct formation at the 4- PP arm (cultured mouse and rat hepatocytes, treated by acetaminophen and ethanol, will be the model in these experiments). (3) Investigate the role of 10-formyl-THF metabolizing pathways in animal models (Aldh1l1, Aldh1l2 and double knockout mice, generated in the PI's lab, will be the model in these experiments). The importance of folate as a nutrient, its well-established role in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer, as well as the necessity for better understanding mechanisms of drug-induce liver injury, the critical role of mitochondria in both folate metabolism and liver injury, nd the growing body of evidence that FDH is a key metabolic regulator make these studies particularly relevant.

描述（由申请人提供）：本提案的主要目标是了解最丰富的叶酸酶之一 10-甲酰四氢叶酸脱氢酶（FDH、ALDH1L1）的代谢作用，以及对其催化的抑制如何参与发病机制。我们之前的研究表明，这种酶存在于许多组织中，但在肝脏和肾脏中含量最丰富，其中肝脏和肾脏的含量高达胞浆蛋白总量的 1.2%。同时，通过其启动子区域的 CpG 岛甲基化，它在人类癌症中被强烈且普遍地下调。此外，与对乙酰氨基酚过量或饮酒相关的酶的抑制被认为会导致肝毒性。 FDH 将 10-甲酰四氢叶酸 (10-fTHF) 转化为四氢叶酸 (THF) 和 CO2。重要的是，该反应以二氧化碳的形式从叶酸库中去除碳基团，从而降低叶酸依赖性生物合成能力。基于此，我们的第一个假设是 FDH 调节单碳基团通过叶酸库的整体流动，并且是控制细胞增殖的内在机制之一。 FDH 反应也是甲酸盐清除的限速步骤。值得注意的是，甲酸是一种有毒代谢物，通常通过多种生理途径在人体中产生。因此，我们的第二个假设是，抑制非增殖肝细胞中的 FDH 将导致甲酸盐毒性。我们最近发现 FDH 需要辅基 4'- 磷酸泛硫氨酸 (4-PP) 进行催化。这种灵活的长臂与酶共价连接，在两个催化位点之间转移反应中间体，其去除/修饰完全“杀死”酶活性。因此，我们的第三个假设是对乙酰氨基酚和乙醇的活性代谢物通过与其辅基形成加合物来抑制FDH活性，并且这种抑制导致肝损伤。重要的是，在上一个资助周期中，我们发现了一种新的酶，线粒体 FDH (ALDH1L2)。在目前的提案中，我们不仅将FDH作为研究目标，还将通过在方程中添加该途径的线粒体分支来探索整个10-fTHF到THF途径的重要性。解决我们的假设的具体目标是：（1）解析全长apo-FDH 和4-PP 修饰的全酶的晶体结构。 (2) 确定FDH是否在体内被4-PP臂上的共价加合物形成所抑制（培养的小鼠和大鼠肝细胞，经对乙酰氨基酚和乙醇处理，将成为这些实验的模型）。 （3）研究10-甲酰基-THF代谢途径在动物模型中的作用（PI实验室制备的Aldh1l1、Aldh1l2和双基因敲除小鼠将作为本次实验的模型）。叶酸作为一种营养素的重要性，其在预防巨幼细胞贫血、血管疾病、神经管先天缺陷和癌症方面的既定作用，以及更好地了解药物引起的肝损伤机制的必要性、线粒体在叶酸代谢和肝损伤中的关键作用，以及越来越多的证据表明 FDH 是关键的代谢调节剂，使这些研究显得尤为重要。