Branched chain acyl-CoA metabolism and disease

支链酰基辅酶A代谢与疾病

基本信息

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
项目类别：
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

项目摘要

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会导致 13 种代谢紊乱但本质上什么都没有了解定义 BCAA 代谢的物理参数，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，并表征了它们的特征酶的功能以及与其缺乏相关的疾病。最近，ACAD10 基因的变异已被证明与皮马人患 2 型糖尿病 (T2DM) 的风险相关印度人，但这种风险的机制尚未阐明。检验 ACAD10 在 T2DM 的发展，我们生成了敲除小鼠模型。引人注目的是，缺乏营养的动物却获得了过多的营养。体重增加并产生胰岛素抵抗。该项目的长期目标是表征支链酰基辅酶A并确定其在人类中失败的后果。本次续签申请具有三个具体目标。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线粒体内的功能复合物。具体目标 1a 是使用免疫和冷冻电子显微镜可视化 BCAA 代谢复合物。具体目标 1b 将采用免疫共沉淀，有或没有化学交联以识别支链氨基酸代谢复合物中的结合伙伴。我预测分支链特异性 ACAD 将与支链酮酸脱氢酶紧密相互作用，形成支链酮酸脱氢酶的核心支链氨基酸代谢复合物。具体目标 1c 是检查遗传中 BCAA 复合物的破坏 ACAD 疾病。我假设不同基因的突变引起的差异效应是导致这些疾病中出现的广泛临床症状的原因。具体目标 2 是证明正常细胞和缺陷细胞中支链氨基酸底物的代谢通道在支链特定的 ACAD 中。具体目标 2a 是利用代谢流研究进一步表征 BCAA 代谢。我预测由标记的奇数链脂肪酸生成的丙酰辅酶A将很容易进入 TCA 循环，而来自标记的 BCAA 则不会。具体目标 2b 是表征缺乏支链 ACAD 之一的细胞中通过 BCAA 的通量。我预测 ILE 和 VAL 的通量 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 IVDH 细胞系中的代谢物。具体目标 3 是描述 T2DM 发展的机制在 ACAD10 缺陷小鼠中。具体目标 3a 是表征 ACAD10 的酶功能。我建议对 ACAD10 小鼠的各种组织进行扩展定向代谢组学研究，以确定候选者酶的底物。具体目标 3b 是表征 ACAD10 缺陷中的葡萄糖胰岛素轴老鼠。这些实验将有助于更好地理解定义缺陷临床风险的机制这些酶。