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：
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
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/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

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.

分支链氨基酸分解代谢的缺陷引起13种代谢性疾病，但实际上没有知道定义BCAA变质的物理参数，或者其破坏与人类疾病。我的实验室定义了许多编码这些蛋白质的基因，并表征了它们酶促功能及其缺乏症相关的疾病。最近，Acad10基因的变体已显示与PIMA中2型糖尿病（T2DM）的发展风险有关印第安人，但这种风险的机制尚未阐明。检查Acad10在 T2DM的开发，我们生成了一个敲除鼠标模型。令人惊讶的是，不足的动物获得了过多的重量并变得耐胰岛素。该项目的远程目标是表征分支链酰基烟草，并确定其在人类失败的后果。此更新应用具有三个具体目标。特定目的1是检查分支链氨基的线粒体结构酸代谢。我假设负责分支链氨基酸代谢的蛋白质形成A 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检查可视化BCAA代谢复合物。具体目标1B将在有和没有的情况下采用共免疫沉淀化学交联以鉴定BCAA代谢复合物中的结合伴侣。我预测分支机构链特异性学院将与分支链酮酸脱氢酶紧密相互作用，以形成 BCAA代谢络合物。特定目标1C是检查遗传中BCAA复合物的破坏学院的疾病。我假设各种基因突变引起的差异效应负责这些疾病中看到的各种临床症状。具体目标2是证明了正常细胞中分支链氨基酸底物的代谢通道和那些不足在分支连锁特定的学院中。具体目标2a是使用代谢通量研究进一步特征BCAA代谢。我预测由标记的奇数链脂肪酸产生的丙酰辅酶A会很容易输入TCA周期，而从标记的BCAA派生则不会。特定目标2B是表征在一个分支链学院中缺乏细胞中通过BCAA的通量。我预测Ile和Val的通量通过SBCAD和IBDH缺陷细胞系中完整途径的代谢物将超过亮氨酸 IVDH细胞系中的代谢产物。特定目的3是表征开发T2DM的机制在Acad10缺乏小鼠中。特定目标3a是表征Acad10的酶促功能。我建议对来自Acad10小鼠的各种组织进行扩展的定向代谢组学研究，以识别候选者酶的底物。特定目标3B是表征Acad10中的葡萄糖胰岛素轴老鼠。这些实验将可以更好地理解定义缺陷临床风险的机制这些酶。