Mechanism of Action of a Major Folate Enzyme

描述(由申请人提供)：这项建议的广泛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，以及其催化抑制如何参与发病机制。我们以前的研究表明，该酶存在于许多组织中，但在肝脏和肾脏中含量最高，前者高达细胞质蛋白总量的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而普遍地下调。此外，与醋氨酚过量或饮酒有关的酶抑制被认为是导致肝脏毒性的原因之一。FDH将10-甲酰基四氢叶酸(10-fTHF)转化为四氢叶酸(THF)和二氧化碳。重要的是，这个反应以二氧化碳的形式从叶酸库中去除了碳基，从而降低了叶酸依赖的生物合成能力。基于此，我们的第一个假设是，FDH调节一碳基团通过叶酸库的整体流动，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，甲酸盐是一种有毒的代谢物，通常通过几种生理途径在人体内产生。因此，我们的第二个假设是，抑制非增殖性肝细胞中的FDH将导致甲酸盐毒性。我们最近发现，外佣需要4‘-磷酸丙氨酸(4-PP)基团来催化。这种灵活的长臂以共价方式连接到酶上，在两个催化位置之间转移反应中间体，它的移除/修饰完全“杀死”了酶的活性。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假体基团形成加合物来抑制FDH的活性，这种抑制作用有助于肝脏损伤。重要的是，在前一个资助周期中，我们发现了一种新的酶，线粒体脱氢酶(ALDH1L2)。在目前的方案中，我们不仅将外佣作为研究对象，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途径的意义。解决我们假设的具体目的是：(1)解决全长apo-FDH和4-PP修饰的全酶的晶体结构。(2)确定在体内是否通过在4-PP臂形成共价加合物来抑制FDH(本实验将以醋氨酚和乙醇处理培养的小鼠和大鼠肝细胞为模型)。(3)研究10-甲酰-四氢呋喃代谢途径在动物模型中的作用(将以PI实验室培育的ALDH1L1、Aldh1L2和双基因敲除小鼠为实验模型)。叶酸作为一种营养素的重要性，其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广泛作用，以及更好地了解药物诱导的肝损伤的机制的必要性，线粒体在叶酸代谢和肝脏损伤中的关键作用，以及越来越多的证据表明FDH是关键的代谢调节因子，使这些研究特别相关。

DESCRIPTION (provided by applicant): The broad objectives of this proposal are to understand the metabolic role of one of the most abundant folate enzymes, 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.