Regulation of mitochondrial metabolism by lysine acetylation

描述（由申请人提供）：我的研究项目的长期目标是了解脂肪酸氧化是如何调节的，以便开发能量代谢疾病的新疗法。初步研究已经确定蛋白质乙酰化/去乙酰化是调节线粒体脂肪酸氧化的新机制。关键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依赖的去乙酰化酶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乙酰化，并且假设它们是sirtuin去乙酰化酶的靶标。特异性目的1将研究线粒体sirtuins 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蛋白（ETF）的乙酰化/去乙酰化也将被研究。据推测，与LCAD类似，这些酶的活性将通过sirtuin去乙酰化来调节。蛋白质组学方法将用于鉴定负责调节酶功能的乙酰化位点。这些位点将进一步研究使用定点诱变和三维分子模型。特异性目标2将关注乙酰化改变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的去乙酰化介导的。我们提出了实验来验证K42的乙酰化通过干扰电子向ETF的结合和转移来降低酶活性的假设。基于酰基辅酶a脱氢酶之间共享的季元结构，该机制有望扩展到其他酶。特异性Aim 3a将在体内研究乙酰化/去乙酰化对LCAD的调节作用，使用转基因小鼠表达Flag标记的LCAD作为报告酶，该报告酶可以很容易地从组织提取物中回收，用于分析乙酰化水平和功能。LCAD-Flag转基因小鼠将与Sirt3敲除小鼠杂交，并在正常和紊乱的代谢状态下进行研究，包括空腹和高脂肪饮食引起的肥胖。我推测LCAD的乙酰化会随着代谢状态的改变而改变，残基K42上Sirt3的活性对于维持体内LCAD的功能是重要的。Specific Aim 3b将使用制备性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。综上所述，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。

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.