Branched chain acyl-CoA metabolism and disease

支链酰基辅酶A代谢与疾病

• 批准号: 9308948
• 负责人: GERARD VOCKLEY
• 金额: $ 46.61万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9308948
• 关键词: Acyl Coenzyme A; Amino Acids; Animals; Architecture; Binding; Biological Assay; Branched-Chain Amino Acids; Catabolism; Cell Line; Cells; Chemicals; Child; Citric Acid Cycle; Clinical; Co-Immunoprecipitations; Collaborations; Complex; Data; Decarboxylation; Defect; Development; Diabetes Mellitus; Disease; Distal; Down-Regulation; Electron Transport; Enzymes; Failure; Fatty Acids; Genes; Glucose; Goals; Grant; Hereditary Disease; Human; Insulin; Insulin Resistance; Isoleucine; Keto Acids; Knockout Mice; Label; Leucine; Mass Spectrum Analysis; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Molecular; Multienzyme Complexes; Mus; Mutation; Neonatal Screening; Non-Insulin-Dependent Diabetes Mellitus; Normal Cell; Obesity; Oxidoreductase; Pathway interactions; Pharmacologic Substance; Pima Indian; Proteins; Reagent; Risk; Role; Signal Transduction; Symptoms; Techniques; Tissues; Valine; Variant; Weight; acyl-CoA dehydrogenase; amino acid metabolism; clinical risk; crosslink; enzyme substrate; experience; experimental study; fatty acid oxidation; glucose metabolism; human disease; in vivo; isoleucylvaline; lipid metabolism; long chain fatty acid; metabolomics; mouse model; novel; organic acid; oxidation; propionyl-coenzyme A; response; tandem mass spectrometry; transamination

Defects in the catabolism of branched chain amino acids cause 13 metabolic disorders Yet essentially nothing is known about the physical parameters defining the BCAA metabolon, or the relationship of its disruption to human disease. My lab has defined many of the genes encoding these proteins, and characterized their enzymatic function and the disorders associated with their deficiency. Recently, variants in the ACAD10 gene have been shown to be associated with a risk for development of type 2 diabetes mellitus (T2DM) in Pima Indians, but the mechanism of this risk has not been elucidated. To examine the role of ACAD10 in the development of T2DM, we generated a knock out mouse model. Strikingly, deficient animals gained excessive weight and became insulin resistant. The long-range goal of this project is to characterize the metabolism of branched chain acyl-CoAs and to identify the consequences of its failure in humans. This renewal application has three specific aims. Specific Aim 1 is to examine the mitochondrial architecture of branched chain amino acid metabolism. I hypothesize that the proteins responsible for branched chain amino acid metabolism form a functional complex within mitochondria. Specific Aim 1a is to use immuno- and cryo-electronmicroscopy to visualize the BCAA metabolic complex. Specific Aim 1b will employ co-immunoprecipitation with and without chemical crosslinking to identify binding partners in the BCAA metabolic complex. I predict that the branched chain specific ACADs will interact tightly with the branched chain keto-acid dehydrogenase to form the core of the BCAA metabolic complex. Specific Aim 1c is to examine disruption of the BCAA complex in genetic disorders of of the ACADs. I hypothesize that differential effects caused by the mutations in the various genes are responsible for the broad array of clinical symptoms seen in these disease. Specific Aim 2 is to demonstrate metabolic channeling of branched chain amino acid substrates in normal cells and those deficient in the branched chain specific ACADs. Specific Aim 2a is to use of metabolic flux studies to further characterize BCAA metabolism. I predict that propionyl-CoA generated from a labeled odd chain fatty acid will readily enter the TCA cycle while that derived from labeled BCAA will not. Specific Aim 2b is to characterize the flux through BCAA in cells deficient in one of the branched chain ACADs. I predict that flux of ILE and VAL metabolites through intact pathways in SBCAD and IBDH deficient cell lines will exceed that of leucine metabolites in IVDH cell lines. Specific Aim 3 is to characterize the mechanisms for the development of T2DM in ACAD10 deficient mice. Specific Aim 3a is to characterize the enzymatic function of ACAD10. I propose to perform expanded directed metabolomics studies on various tissues from ACAD10 mice to identify candidate substrates for the enzyme. Specific Aim 3b is to characterize the glucose insulin axis in ACAD10 deficient mice. These experiments will allow a better understanding of the mechanisms that define clinical risk in defects of these enzymes.

支链氨基酸分解代谢缺陷导致13种代谢紊乱，但基本上没有 已知定义支链氨基酸代谢物的物理参数，或其破坏与 人类疾病。我的实验室已经定义了许多编码这些蛋白质的基因，并表征了它们的 酶的功能和与其缺乏相关的疾病。最近，ACAD10基因的变体 已被证明与患2型糖尿病(T2 DM)的风险有关 印度人，但这种风险的机制尚未阐明。研究ACAD10在人类免疫系统中的作用 发展T2 DM，我们产生了一个基因敲除的小鼠模型。令人惊讶的是，缺乏营养的动物长得过多 体重增加并对胰岛素产生抵抗力。这个项目的长期目标是描述新陈代谢 支链酰基-COAS，并确定其在人类中失效的后果。此续期申请 有三个具体目标。具体目的1是研究支链氨基的线粒体结构。 酸代谢。我假设负责支链氨基酸新陈代谢的蛋白质形成一个 线粒体内的功能复合体。具体目标1a是使用免疫和冷冻电子显微镜 想象支链氨基酸代谢复合体。特殊目标1b将在有和没有的情况下采用免疫共沉淀 化学交联以识别支链氨基酸代谢复合体中的结合伙伴。我预测分枝的 链特异的AADS将与支链酮酸脱氢酶紧密相互作用，形成 支链氨基酸代谢复合体。具体目标1c是检查基因中支链氨基酸复合体的破坏 ACADS的紊乱。我假设不同基因的突变所引起的差异效应 对这些疾病中出现的一系列临床症状负有责任。具体目标2是 证明支链氨基酸底物在正常细胞和缺陷细胞中的代谢通道 在支链中特定的ACADS。具体目标2a是利用代谢流研究进一步 描述支链氨基酸的代谢。我预测由标记的奇链脂肪酸生成的丙酰辅酶A将 很容易进入TCA循环，而从标记的BCAA衍生的循环则不会。具体目标2b是描述 支链乙酰胆碱脱氢酶缺乏的细胞中通过支链氨基酸的通量。我预测Ile和Val的流动 SBCAD和IBDH缺陷细胞系通过完整途径的代谢物将超过亮氨酸 IVDH细胞系中的代谢物。具体目标3是描述T2 DM的发生机制 在ACAD10缺陷小鼠中。具体目的3a是研究ACAD10的酶功能。我提议 对ACAD10小鼠不同组织进行扩展定向代谢组学研究以确定候选 酶的底物。特异靶3b是表征ACAD10缺陷患者的葡萄糖-胰岛素轴 老鼠。这些实验将使我们能够更好地理解定义缺陷临床风险的机制。 这些酶中。