Inborn Errors of Long Chain Fat Metabolism

描述(由申请人提供)： 酰辅酶A脱氢酶(ACDS)是一个多聚黄酮酶家族，催化脂肪酸2-氧化和氨基酸分解代谢中酰基辅酶A酯的1，2-脱氢反应。已在七种ACD中发现了先天新陈代谢缺陷。这个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。我们已经描述和描述了ACD基因家族的几个新成员。在这些酶中，有3种具有显著活性的长链底物：长链和超长链酰辅酶A脱氢酶(分别为LCAD和VLCAD)，以及ACD9和。我们前期和初步的研究表明，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。这一修订应用的目标是表征LCAD、VLCAD和ACD9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。具体目的1是描述超长链酰辅酶A脱氢酶(VLCAD)的变异形式以及这种疾病临床变异的分子基础。特异性目的1a是确定VLCAD在线粒体膜上的独特定位中起重要作用的氨基酸基序(S)。具体目的1b是确定体内发现的VLCAD的替代形式。我们已经在体内鉴定了这种酶的三种不同形式，它们是通过选择性剪接产生的。我假设每一个都有不同的底物专一性，为逐渐变短的底物种类提供功能优化。具体目的1c是研究VLCAD患者突变对酶功能的影响。具体目标2是更全面地描述ACD9及其在人类中的缺陷。具体目标2a是识别其他ACD9缺乏症患者并定义其临床谱系。具体目的2b是研究ACD9的亚细胞分布以及ACD9蛋白在线粒体外的功能和分子结构。我推测，这种替代形式的ACD9在细胞中具有非酶“兼职”功能。具体目的3是阐明LCAD的生理功能。尽管它很早就被认识到了，但它在体内的代谢作用仍然是一个谜。我们的初步数据表明，它与胆汁酸和表面活性物质的代谢有关。具体目的3a是研究LCAD在胆汁酸合成中的作用。我推测它是鹅去氧胆酸合成的关键中间步骤，在线粒体的酸性途径中参与了细胞代谢率的控制。具体目的3b是利用LCAD缺失小鼠探索LCAD在表面活性物质代谢中的作用。这些研究需要从根本上改变我们对线粒体2-氧化的看法，从只负责能量产生的代谢途径转变为在其他重要生物过程中也活跃于以前未被识别的各种功能的代谢途径。

DESCRIPTION (provided by applicant): 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 this revised 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 1 is to characterize variant forms of very long chain acyl-CoA dehydrogenase (VLCAD) and the molecular basis of clinical variability in this disorder. Specific aim 1a is to identify the amino acid motif(s) important in determining the unique localization of VLCAD to the inner mitochondrial membrane. Specific aim 1b is to characterize alternative forms of VLCAD identified in vivo. We have identified 3 variant forms of this enzyme in vivo that are generated through alternative splicing. I hypothesize that each has a different substrate specificity that provides functional optimization for progressively shorter substrate species. Specific aim 1c is to characterize the effect of patient mutations in VLCAD on enzyme function. Specific Aim 2 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. Specific Aim 3 is to elucidate the physiologic function of LCAD. Despite its early recognition, its in vivo metabolic role remains a mystery. Our preliminary data implicates it in bile acid and surfactant metabolism. Specific Aim 3a is to characterize the role of LCAD in bile acid synthesis. I hypothesize that it characterizes a key intermediate step in chenodeoxycholic acid synthesis in a mitochondrial based acidic pathway that is involved in the control of cellular metabolic rate. Specific Aim 3b is to explore the role of LCAD in surfactant metabolism using an LCAD null mouse. These studies necessitate a fundamental revision in our view of mitochondrial 2-oxidation from a metabolic pathway that is only responsible for energy generation to one that is active as well in a variety of previously unrec- ognized functions in other important biologic processes.