Chemical Acetylation of Mitochondrial Fatty Acid Oxidation Enzymes by Acetyl-CoA

描述(由申请人提供)： 申请者研究计划的长期目标是了解负责控制线粒体脂肪酸新陈代谢的调节机制。一个为期两年的项目被提出，以研究乙酰辅酶A脱氢酶(ACAD)家族成员上赖氨酸残基的乙酰化。AADS是线粒体黄素酶，催化脂肪酸氧化的第一步。这些酶的遗传缺陷是新陈代谢最常见的先天缺陷之一。此外，肥胖、糖尿病和其他常见疾病都与功能失调的线粒体脂肪酸氧化有关。因为它们催化脂肪酸氧化循环中的限速步骤，所以调节AADS的因子调节脂肪酸产生能量的速率。初步数据表明，通过赖氨酸乙酰化的翻译后修饰可以对长链酰辅酶A脱氢酶(LCAD)的活性进行两倍的调节。LCAD和其他线粒体蛋白质乙酰化的机制尚不清楚。虽然赖氨酸乙酰转移酶存在于细胞核和其他细胞内隔室，但在线粒体中没有发现。初步数据显示，几种ACAD酶的乙酰化可以发生在化学上。纯化的重组Acads与浓度大于0.5 mM的乙酰辅酶A孵育，导致赖氨酸乙酰化。由于线粒体乙酰辅酶A浓度可能达到2 mM或更高，推测化学乙酰化是肝脏、肌肉、心脏和棕色脂肪等高度氧化组织线粒体中赖氨酸乙酰化现象的原因。提出了研究这一现象的两个目标。目的1表征ACAD酶的非酶赖氨酸乙酰化反应。这包括对反应条件、乙酰转移酶抑制剂的使用以及检测和定量赖氨酸乙酰化的多种方法的全面调查。目的2是确定ACAD蛋白表面微环境对赖氨酸乙酰化的影响。基于三维分子模拟和其他初步数据，推测位于酸性氨基酸附近的赖氨酸由于酸性残基的活性去质子化而特别容易被修饰。通过与乙酰辅酶A反应而乙酰化的重组AAds将进行质谱分析以确定乙酰化位点。然后，这些位置将使用分子建模软件和之前发表的ACAD晶体结构在三维空间中绘制地图。最后，定点突变将被用来确定相邻氨基酸对每个确定的赖氨酸的乙酰化的贡献。拟议的R03项目将与申请者目前的K01职业发展奖产生协同作用，从而更快地过渡到独立和更具竞争力的未来R01申请。 公共健康相关性：线粒体能量代谢的公共健康相关性变化是许多重大疾病(从癌症到糖尿病)的基础，也被认为是衰老过程的核心。这项拟议的研究试图阐明被称为赖氨酸乙酰化的代谢酶修饰背后的分子事件。了解线粒体酶的赖氨酸乙酰化可能会导致开发控制新陈代谢的新药，从而预防疾病，甚至可能减缓衰老。

DESCRIPTION (provided by applicant): The long-term objective of the applicant's research program is to understand regulatory mechanisms responsible for controlling mitochondrial fatty acid metabolism. A two-year project is proposed to investigate acetylation of lysine residues on members of the acyl-CoA dehydrogenase (ACAD) family of proteins. ACADs are mitochondrial flavoenzymes that catalyze the first step in fatty acid oxidation. Genetic deficiencies of these enzymes are among the most common inborn errors of metabolism. Additionally, obesity, diabetes, and other common diseases have been linked to dysfunctional mitochondrial fatty acid oxidation. Because they catalyze the rate-limiting step in the fatty acid oxidation cycle, factors that regulate ACADs regulate the rate of energy production from fatty acids. Preliminary data demonstrate that post-translational modification by lysine acetylation can regulate activity of long- chain acyl-CoA dehydrogenase (LCAD) by two-fold. The mechanism by which LCAD and other mitochondrial proteins become acetylated is not known. While lysine acetyltransferases are present in the nucleus and other intracellular compartments, none have been identified in mitochondria. Preliminary data show that acetylation of several ACAD enzymes can occur chemically. Incubation of purified recombinant ACADs with acetyl-CoA at concentrations greater than 0.5 mM resulted in lysine acetylation. Since mitochondrial acetyl-CoA concentrations may reach 2 mM or higher it is hypothesized that chemical acetylation is responsible for the phenomenon of lysine acetylation seen in mitochondria of highly oxidative tissues such as liver, muscle, heart and brown fat. Two aims are proposed to study the phenomenon. Aim 1 is to characterize non-enzymatic lysine acetylation of ACAD enzymes. This includes a comprehensive survey of reaction conditions, the use of acetyltransferase inhibitors, and multiple methods of detecting and quantifying lysine acetylation. Aim 2 is to determine how microenvironments on the surface of ACAD proteins influence lysine acetylation. Based on three-dimensional molecular modeling and other preliminary data it is postulated that lysines located near acidic amino acids are particularly prone to modification due to active deprotonation by the acidic residues. Recombinant ACADs acetylated by reaction with acetyl-CoA will be subjected to mass spectrometry to identify the acetylation sites. These sites will then be mapped in three-dimensional space using molecular modeling software and previously published ACAD crystal structures. Finally, site-directed mutagenesis will be used to determine the contribution of neighboring amino acids to the acetylation of each identified lysine. The proposed R03 project would synergize with the applicant's current K01 Career Development Award resulting in a more rapid transition to independence and a more competitive future R01 application. PUBLIC HEALTH RELEVANCE: Public Health Relevance Changes in mitochondrial energy metabolism underlie many major diseases from cancer to diabetes, and are also believed to be at the core of the aging process. The proposed research seeks to elucidate the molecular events behind a modification to metabolic enzymes known as lysine acetylation. Understanding lysine acetylation of mitochondrial enzymes may lead to the development of new drugs to control metabolism, thereby preventing disease and perhaps even slowing aging.