Inborn Errors of Long Chain Fat Metabolism

描述（由申请人提供）：本申请涉及通知编号（NOT-OD-09-058）和通知标题：NIH宣布恢复法案资金可用于竞争性修订申请，并要求对R 01 DK 78755进行竞争性修订。酰基辅酶A脱氢酶（acyl-CoA disulfase，ACD）是一类多聚体黄素酶，在脂肪酸2-氧化和氨基酸催化中催化酰基辅酶A酯的1，2-脱氢。在7例ACD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。我们已经描述并表征了ACD基因家族的几个新成员。在这些酶中，有3种酶对长链底物具有显著活性：长链和极长链酰基辅酶A脱氢酶（分别为LCAD和VLCAD），以及ACD 9和ACD 9。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，并存在于多个亚细胞位置。资助申请的目标是表征LCAD，VLCAD和ACD 9的生理作用，并探索这些酶在人类和小鼠模型中遗传缺陷的后果。原始申请的具体目标2是更完整地表征ACD 9及其在人类中的缺陷。具体目标2a是识别其他ACD 9缺陷患者并定义其临床谱。具体目标2b是表征ACD 9的亚细胞分布以及线粒体外ACD 9蛋白的功能和分子构型。我假设这种替代形式的ACD 9在细胞中具有非酶的“兼职”功能。我请求对申请进行补充，以扩展我实现这些目标的能力。首先，我发现了比最初预期更多的潜在缺陷患者。我正在申请资金，以支持额外的技术人员时间，以更快地处理这些样品，加快我们的进度。其次，我们对ACAD 9缺陷的病理生理学的理解和我们研究该基因产物的兼职功能的能力受到仅具有ACAD 9缺陷的组织培养细胞的可用性的限制。为了加强这些研究，我请求补充资金，从德克萨斯A&M基因组医学研究所购买一只ACAD 9基因敲除小鼠，该研究所的目录中有它。将对动物的生化和病理生理表型进行表征，包括代谢特征分析、完整的组织学检查以及基础和静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级分离研究将检查非线粒体位置中ACAD 9的存在和敲除小鼠模型中的缺失。 公共卫生相关性：酰基辅酶A酶是维持体内正常化学平衡的重要酶。我们已经确定了一种新的遗传疾病的这些酶之一，导致肝功能衰竭。研究这种疾病对于了解更多关于其临床表现和治疗非常重要

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