Regulation of mitochondrial metabolism by lysine acetylation

赖氨酸乙酰化调节线粒体代谢

基本信息

批准号：
8489291
负责人：
ERIC S GOETZMAN
金额：
$ 31.28万
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：
2011
资助国家：
美国
起止时间：
2011-06-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8489291
关键词：
Acetylation Acyl CoA Dehydrogenases Binding Biochemical Genetics Biological Assay Brown Fat Cell Culture Techniques Cells Complex Data Deacetylase Deacetylation Development Diabetes Mellitus Diet Disease Electron Transport Electron transfer flavoprotein Energy Metabolism Enzyme Kinetics Enzymes Etiology Event Family Family member Fasting Fatty Acids Fatty acid glycerol esters Functional disorder Genes Genetic Goals Histocompatibility Testing Human Inborn Errors of Metabolism Isoelectric Focusing Isovaleryl-CoA dehydrogenase Knockout Mice Life Liver Long-Chain-Acyl-CoA Dehydrogenase Lysine Malignant Neoplasms Measures Mediating Metabolic Metabolism Methods Mitochondria Mitochondrial Proteins Modification Molecular Molecular Models Mus Mutation Myocardium Neonatal Screening Obesity Organ Oxidants Oxidation-Reduction Pathway interactions Patients Physiological Play Preparation Process Production Protein Acetylation Protein Isoforms Proteins Proteomics Regulation Relative (related person)Reporter Research Role Sirtuins Site Site-Directed Mutagenesis Solutions Structure Testing Tissue Extracts Tissues Transgenic Mice Western Blotting acyl-CoA dehydrogenase base enzyme activity fatty acid metabolism fatty acid oxidation feeding human disease in vitro activity in vivo member molecular modeling mouse model novel programs protein protein interaction research study response

Acetylation Acyl CoA Dehydrogenases Binding Biochemical Genetics Biological Assay Brown Fat Cell Culture Techniques Cells Complex Data Deacetylase Deacetylation Development Diabetes Mellitus Diet Disease Electron Transport Electron transfer flavoprotein Energy Metabolism Enzyme Kinetics Enzymes Etiology Event Family Family member Fasting Fatty Acids Fatty acid glycerol esters Functional disorder Genes Genetic Goals Histocompatibility Testing Human Inborn Errors of Metabolism Isoelectric Focusing Isovaleryl-CoA dehydrogenase Knockout Mice Life Liver Long-Chain-Acyl-CoA Dehydrogenase Lysine Malignant Neoplasms Measures Mediating Metabolic Metabolism Methods Mitochondria Mitochondrial Proteins Modification Molecular Molecular Models Mus Mutation Myocardium Neonatal Screening Obesity Organ Oxidants Oxidation-Reduction Pathway interactions Patients Physiological Play Preparation Process Production Protein Acetylation Protein Isoforms Proteins Proteomics Regulation Relative (related person)Reporter Research Role Sirtuins Site Site-Directed Mutagenesis Solutions Structure Testing Tissue Extracts Tissues Transgenic Mice Western Blotting acyl-CoA dehydrogenase base enzyme activity fatty acid metabolism fatty acid oxidation feeding human disease in vitro activity in vivo member molecular modeling mouse model novel programs protein protein interaction research study response

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项目摘要

DESCRIPTION (provided by applicant): The long term goal of my research program is to understand how fatty acid oxidation is regulated in order to develop new therapies for diseases of energy metabolism. Preliminary studies have identified protein acetylation/deacetylation as a novel mechanism regulating mitochondrial fatty acid oxidation. The key fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) has eight lysine acetylation sites and is demonstrated to be a target of the mitochondrial NAD-dependent deacetylase sirtuin-3 (Sirt3). When co- expressed with Sirt3 in HEK-293 cells, LCAD shows reduced lysine acetylation which is associated with a doubling of enzymatic activity. Other members of the acyl-CoA dehydrogenase enzyme family are also acetylated on numerous lysines and it is hypothesized that they are targets for sirtuin deacetylases. Specific Aim 1 will investigate interactions between the mitochondrial sirtuins Sirt3, Sirt4, and Sirt5 and the enzymes very long-chain acyl-CoA dehydrogenase (VLCAD), medium chain-acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD). Acetylation/deacetylation of their redox partner electron transferring flavoprotein (ETF) will also be studied. It is hypothesized that, similar to LCAD, activity of these enzymes will be modulated by sirtuin deacetylation. Proteomics methods will be used to identify acetylation sites responsible for regulating enzyme function. These sites will be further investigated using site-directed mutagenesis and three-dimensional molecular modeling. Specific Aim 2 will focus on the mechanism by which acetylation alters LCAD activity. Preliminary data suggest that the effect of Sirt3 on LCAD activity is mediated by deacetylation of residue K42. Experiments are proposed to test the hypothesis that acetylation at K42 reduces enzymatic activity by interfering with the binding and transfer of electrons to ETF. Based on a shared quaternary structure among the acyl-CoA dehydrogenases the mechanism is anticipated to extend to other enzymes. Specific Aim 3a will study regulation of LCAD by acetylation/deacetylation in vivo using transgenic mice that express Flag- tagged LCAD as a reporter enzyme that can be easily recovered from tissue extracts for analysis of acetylation levels and function. LCAD-Flag transgenic mice will be crossed with Sirt3 knockout mice and studied under normal versus perturbed metabolic states including fasting and high-fat diet-induced obesity. I hypothesize that acetylation of LCAD will change with metabolic state and that Sirt3 activity on residue K42 is important for maintaining LCAD function in vivo. Specific Aim 3b will use preparative isoelectric focusing to separate differentially acetylated VLCAD, MCAD, IVD and ETF isoforms from mouse liver. Acetylation and enzyme function will be evaluated in protein preparations from Sirt3-/- mice versus wildtype. In summary, it is expected that this project will fundamentally alter our understanding of how acyl-CoA dehydrogenases and fatty acid oxidation are regulated and will uncover important new targets for treating diseases of energy metabolism.

描述（由申请人提供）：我的研究计划的长期目标是了解如何调节脂肪酸氧化，以开发能量代谢疾病的新疗法。初步研究已将蛋白质乙酰化/脱乙酰化确定为调节线粒体脂肪酸氧化的新机制。钥匙脂肪酸氧化酶长链酰基-COA脱氢酶（LCAD）具有八个赖氨酸乙酰化位点，并且被证明是线粒体NAD依赖性脱乙酰基二乙烯基sirtuin-3（SIRT3）的靶标。当在HEK-293细胞中与SIRT3共同表达时，LCAD显示赖氨酸乙酰化降低，这与酶活性的加倍有关。酰基-COA脱氢酶家族的其他成员在许多赖氨酸上也被乙酰化，并且假设它们是西二素脱乙酰基酶的靶标。具体目标1将研究线粒体SIRTUINS SIRT3，SIRT4和SIRT5与非常长链酰基酰基-COA脱氢酶（VLCAD），中链 - 酰基-COAA脱氢酶（MCAD）和Isovaleryl-COA-COA脱氢酶（IVD）之间的相互作用。还将研究其氧化还原伴侣电子转移黄蛋蛋白（ETF）的乙酰化/脱乙酰化。假设与LCAD相似，这些酶的活性将通过脱乙酰基化调节。蛋白质组学方法将用于识别负责调节酶功能的乙酰化位点。这些位点将使用位置定向的诱变和三维分子建模进一步研究。具体目标2将重点放在乙酰化改变LCAD活性的机制上。初步数据表明，SIRT3对LCAD活性的影响是由残基K42的脱乙酰化介导的。提出了实验来检验以下假设：K42处的乙酰化通过干扰电子与ETF的结合和转移来降低酶促活性。基于酰基-COA脱氢酶中共有的四元结构，预计机理将延伸至其他酶。特定的目标3a将使用乙酰化/脱乙酰化在体内研究LCAD，使用转基因小鼠表达标记为标记的LCAD作为报告酶，可以轻松地从组织提取物中恢复，以分析乙酰化水平和功能。 LCAD-FLAG转基因小鼠将与SIRT3基因敲除小鼠交叉，并在正常的与扰动的代谢状态下进行研究，包括禁食和高脂饮食诱导的肥胖症。我假设LCAD的乙酰化将随代谢状态而变化，而在残基K42上的SIRT3活性对于维持体内LCAD功能很重要。特定的目标3B将使用制备异型聚焦来分离乙酰化的VLCAD，MCAD，IVD和ETF同工型与小鼠肝脏。乙酰化和酶功能将在SIRT3 - / - 小鼠与野生型的蛋白质制剂中评估。总而言之，预计该项目将从根本上改变我们对酰基-COA脱氢酶和脂肪酸氧化如何受到调节的理解，并将发现重要的新靶标以治疗能量代谢疾病。