Branched chain acyl-CoA metabolism and disease

支链酰基辅酶A代谢与疾病

• 批准号: 9130361
• 负责人: GERARD VOCKLEY
• 金额: $ 46.16万
• 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/9130361
• 关键词: Acyl CoA Dehydrogenases; 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; fatty acid oxidation; glucose metabolism; human disease; in vivo; lipid metabolism; long chain fatty acid; metabolomics; mouse model; novel; organic acid; propionyl-coenzyme A; research study; 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.

支链氨基酸催化剂的缺陷导致13种代谢紊乱，但基本上没有什么 已知BCAA代谢子的物理参数，或其破坏与 人类疾病我的实验室已经确定了许多编码这些蛋白质的基因，并描述了它们的特征。 酶功能和与其缺乏相关的疾病。最近，ACAD10基因的变异 已被证明与皮马2型糖尿病（T2DM）的发生风险相关 印度人，但这种风险的机制尚未阐明。研究ACAD10在 在T2DM的发展中，我们产生了敲除小鼠模型。令人惊讶的是，缺乏的动物获得了过量的 体重增加，并出现胰岛素抵抗。该项目的长期目标是描述 支链酰基辅酶A，并确定其在人类中失败的后果。这份更新申请 有三个具体目标。具体目标1是检查支链氨基的线粒体结构， 酸代谢我假设负责支链氨基酸代谢的蛋白质形成了一个 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， 将BCAA代谢复合物可视化。Specific Aim 1b将采用免疫共沉淀， 化学交联以鉴定BCAA代谢复合物中的结合伴侣。我预测， 链特异性ACADs将与支链酮酸脱氢酶紧密地相互作用，以形成 BCAA代谢复合物。具体目标1c是检查BCAA复合物在遗传学中的破坏， ACADs的紊乱。我假设不同基因突变引起的不同效应 是导致这些疾病中广泛临床症状的原因。具体目标二是 证明了正常细胞和那些缺陷细胞中支链氨基酸底物的代谢通道 在支链特异性ACADs中。具体目标2a是利用代谢通量研究进一步 表征支链氨基酸代谢。我预测，从标记的奇数链脂肪酸产生的丙酰辅酶A将 容易进入TCA循环，而来自标记BCAA的则不会。具体目标2b是表征 在缺乏支链ACADs之一的细胞中通过BCAA的流量。我预测ILE和瓦尔的通量 在SBCAD和IBDH缺陷细胞系中通过完整途径的代谢物将超过亮氨酸的代谢物。 IVDH细胞系中的代谢物。具体目标3是表征T2DM发生的机制 在ACAD10缺陷小鼠中。具体目标3a是表征ACAD10的酶功能。我建议 对来自ACAD10小鼠的各种组织进行扩展定向代谢组学研究，以鉴定候选物 酶的底物。具体目标3b是表征ACAD10缺陷型中的葡萄糖胰岛素轴。 小鼠这些实验将使我们更好地理解缺陷的临床风险机制 这些酶。