Regulation of mitochondrial metabolism by lysine acetylation

赖氨酸乙酰化调节线粒体代谢

• 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
• 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

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，以便开发治疗能量代谢疾病的新疗法。初步研究已确定蛋白质乙酰化/脱乙酰化是调节线粒体脂肪酸氧化的新机制。关键的脂肪酸氧化酶长链酰基辅酶 A 脱氢酶 (LCAD) 具有 8 个赖氨酸乙酰化位点，并被证明是线粒体 NAD 依赖性脱乙酰酶 Sirtuin-3 (Sirt3) 的靶标。当在 HEK-293 细胞中与 Sirt3 共表达时，LCAD 显示赖氨酸乙酰化减少，这与酶活性加倍相关。酰基辅酶A脱氢酶家族的其他成员也在许多赖氨酸上被乙酰化，并且假设它们是sirtuin脱乙酰酶的靶标。具体目标 1 将研究线粒体去乙酰化酶 Sirt3、Sirt4 和 Sirt5 与超长链酰基辅酶 A 脱氢酶 (VLCAD)、中链酰基辅酶 A 脱氢酶 (MCAD) 和异戊酰基辅酶 A 脱氢酶 (IVD) 之间的相互作用。还将研究其氧化还原伙伴电子转移黄素蛋白 (ETF) 的乙酰化/脱乙酰化。据推测，与 LCAD 类似，这些酶的活性将受到沉默调节蛋白脱乙酰化的调节。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。将使用定点诱变和三维分子模型进一步研究这些位点。具体目标 2 将重点关注乙酰化改变 LCAD 活性的机制。初步数据表明，Sirt3 对 LCAD 活性的影响是通过残基 K42 的脱乙酰化介导的。提出实验来检验 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的假设。基于酰基辅酶A脱氢酶之间共享的四级结构，预计该机制将扩展到其他酶。具体目标 3a 将使用转基因小鼠研究体内乙酰化/脱乙酰化对 LCAD 的调节，这些转基因小鼠表达带标记的 LCAD 作为报告酶，可以轻松地从组织提取物中回收该报告酶，以分析乙酰化水平和功能。 LCAD-Flag 转基因小鼠将与 Sirt3 敲除小鼠杂交，并在正常代谢状态与紊乱代谢状态（包括禁食和高脂肪饮食诱导的肥胖）下进行研究。我假设 LCAD 的乙酰化会随着代谢状态而改变，并且残基 K42 上的 Sirt3 活性对于维持体内 LCAD 功能很重要。 Specific Aim 3b 将使用制备型等电聚焦从小鼠肝脏中分离差异乙酰化的 VLCAD、MCAD、IVD 和 ETF 亚型。将在 Sirt3-/- 小鼠与野生型小鼠的蛋白质制剂中评估乙酰化和酶功能。总之，预计该项目将从根本上改变我们对酰基辅酶A脱氢酶和脂肪酸氧化如何调节的理解，并将发现治疗能量代谢疾病的重要新靶点。