Inborn Errors of Long Chain Fat Metabolism

描述（由申请人提供）：本申请地址通知号（NOT-OD-09-058）和通知标题：NIH宣布为竞争性修订申请提供恢复法案资金，并要求对R01DK78755进行竞争性修订。酰基辅酶a脱氢酶（ACDs）是一个多聚黄酶家族，在脂肪酸2氧化和氨基酸分解代谢中催化酰基辅酶a酯的1,2-脱氢。在7例ACDs中发现了先天性代谢缺陷。该项目的长期目标是研究ACD基因家族中重要的结构/功能关系。我们已经描述和表征了ACD基因家族的几个新成员。其中有3种酶对长链底物具有显著的活性：长链酰基辅酶a脱氢酶和超长链酰基辅酶a脱氢酶（分别为LCAD和VLCAD），以及ACD9和ACD9。我们先前和初步的研究表明，这些酶具有不同的底物利用谱，组织和发育表达模式，在细胞中以多种活性形式存在，并且存在于多个亚细胞位置。该资助申请的目标是表征LCAD， VLCAD和ACD9的生理作用，并探索人类和小鼠模型中这些酶的遗传缺陷的后果。最初应用的具体目的2是更完整地表征ACD9及其在人类中的缺陷。具体目标2a是识别更多的ACD9缺乏症患者并定义其临床谱。特异性Aim 2b是表征ACD9的亚细胞分布以及线粒体外ACD9蛋白的功能和分子构型。我假设这种替代形式的ACD9在细胞中具有非酶的“兼职”功能。我要求一份补充申请，以扩大我的能力，以满足这些目标。首先，我发现了比原先预期更多的潜在缺陷患者。我请求资金支持额外的技术人员时间来更快地处理这些样品，加快我们的进展。其次，我们对ACAD9缺乏症的病理生理学的理解和研究该基因产物的兼职功能的能力受到仅有ACAD9缺乏症的组织培养细胞的限制。为了加强这些研究，我正在申请补充资金，从德克萨斯农工大学基因组医学研究所购买一只ACAD9基因敲除小鼠，该研究所的目录中有这种小鼠。动物的生化和病理生理表型将被表征，包括代谢谱，完整的组织学调查，以及在基础和休息条件下缺乏对动物的生理影响。免疫组织化学和亚细胞分离研究将检查ACAD9在非线粒体位置的存在和敲除小鼠模型中的丢失。

DESCRIPTION (provided by applicant): This application addresses Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications, and asks for competitive revision of R01DK78755. The acyl-CoA dehydrogenases (ACDs) are a family of multimeric flavoenzymes that catalyze the 1,2-dehydrogenation of acyl-CoA esters in fatty acid 2-oxidation and amino acid catabolism. Inborn errors of metabolism have been identified in seven of the ACDs. The long range objective of this project has been to investigate important structure/function relationships in the ACD gene family. We have described and characterized several new members of the ACD gene family. Among these are 3 enzymes with significant activities with long chain substrates: long and very long chain acyl-CoA dehydrogenases (LCAD and VLCAD, respectively), and ACD9 and. Our prior and preliminary studies show that these enzymes have distinct substrate utilization profiles, tissue and developmental expression patterns, exist in multiple active forms in the cell, and are present in multiple subcellular locations. The goal of the funded application is to characterize the physiologic roles of LCAD, VLCAD, and ACD9 and explore the ramifications of genetic deficiencies of these enzymes in humans and mouse models. Specific Aim 2 of the original application is to more completely characterize ACD9 and its deficiency in humans. Specific Aim 2a is identification of additional patients with ACD9 deficiency and definition of its clinical spectrum. Specific Aim 2b is characterization of the subcellular distribution of ACD9 and the function and molecular configuration of ACD9 protein outside of mitochondria. I hypothesize that this alternative form of ACD9 has non-enzymatic "moonlighting" functions in the cell. I am requesting a supplement to the application to extend my ability to address these aims. First, I have identified more potential deficient patients than originally anticipated. I am requesting funds to support additional technician time to process these samples more quickly and accelerate our progress. Secondly, our understanding of the pathophysiology of ACAD9 deficiency and our ability to study the moonlighting functions of this gene product are limited by the availability of only tissue culture cells with ACAD9 deficiency. To augment these studies, I am requesting supplemental funds to purchase an ACAD9 knock out mouse from the Texas A&M Institute for Genome Medicine, which has it available in its catalogue. The biochemical and pathophysiologic phenotype of the animal will be characterized including metabolic profiling, complete histologic survey, and physiologic effects of the deficiency on the animal under basal and resting conditions. Immunohistochemistry and subcellular fractionation studies will examine for presences of ACAD9 in non-mitochondrial locations and loss in the knock out mouse model. PUBLIC HEALTH RELEVANCE: The acyl-CoA dehydrogenases are important enzymes in maintaining normal chemical balance in the body. We have identified a new genetic disorder of one of these enzymes that leads to liver failure. Studying this disorder is important to learn more about its clinical presentation and treatment