Regulation of mitochondrial metabolism by lysine acetylation

赖氨酸乙酰化调节线粒体代谢

• 批准号: 8280418
• 负责人: ERIC S GOETZMAN
• 金额: $ 32.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 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/8280418
• 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

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脂肪酸氧化是如何调节的，以便为能量代谢疾病开发新的治疗方法。初步研究表明，蛋白质乙酰化/去乙酰化是调节线粒体脂肪酸氧化的一种新机制。脂肪酸氧化关键酶长链酰辅酶A脱氢酶(LCAD)有8个赖氨酸乙酰化位点，是线粒体NAD依赖的脱乙酰酶sirtuin-3(SIRT3)的靶标。当与SIRT3在HEK-293细胞中共表达时，LCAD显示赖氨酸乙酰化减少，这与酶活性加倍有关。乙酰辅酶A脱氢酶家族的其他成员也在许多赖氨酸上发生乙酰化，推测它们是sirtuin脱乙酰酶的靶标。具体目标1将研究线粒体sirtuins SIRT3、SIRT4和SIRT5与超长链酰辅酶A脱氢酶(VLCAD)、中链酰辅酶A脱氢酶(MCAD)和异戊酰辅酶A脱氢酶(IVD)之间的相互作用。他们的氧化还原伙伴电子转移黄素蛋白(ETF)的乙酰化/去乙酰化也将被研究。假设，与LCAD类似，这些酶的活性将受到sirtuin脱乙酰基的调节。蛋白质组学方法将被用来确定负责调节酶功能的乙酰化位点。我们将利用定点突变和三维分子模型进一步研究这些位点。具体目标2将集中在乙酰化改变LCAD活性的机制上。初步数据表明，SIRT3对LCAD活性的影响是通过K42残基的脱乙酰基介导的。有人提议通过实验来检验这一假设，即K42处的乙酰化通过干扰电子与ETF的结合和转移来降低酶活性。基于酰基辅酶A脱氢酶之间共享的四元结构，该机制有望扩展到其他酶。具体目标3a将在体内研究乙酰化/去乙酰化对LCAD的调节，使用表达Flag标记的LCAD作为报告酶的转基因小鼠，这种报告酶可以很容易地从组织提取液中回收，用于分析乙酰化水平和功能。LCAD-Flag转基因小鼠将与SIRT3基因敲除小鼠杂交，在正常代谢状态和扰动代谢状态下进行研究，包括禁食和高脂饮食诱导的肥胖。我假设LCAD的乙酰化会随着代谢状态的变化而变化，K42残基上的SIRT3活性对于维持体内LCAD的功能是重要的。利用制备性等电聚焦技术从小鼠肝脏中分离差异乙酰化的VLCAD、MCAD、IVD和ETF亚型。乙酰化和酶功能将在SIRT3-/-小鼠和野生型小鼠的蛋白质制剂中进行评估。总之，预计这个项目将从根本上改变我们对酰辅酶A脱氢酶和脂肪酸氧化是如何调节的理解，并将发现治疗能量代谢疾病的重要新靶点。