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

乙酰辅酶A对线粒体脂肪酸氧化酶的化学乙酰化

• 批准号: 8084081
• 负责人: ERIC S GOETZMAN
• 金额: $ 7.35万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8084081
• 关键词: Acetyl Coenzyme A; Acetylation; Acetyltransferase; Acidic Amino Acids; Acyl CoA Dehydrogenases; Aging; Aging-Related Process; Amino Acids; Brown Fat; Cell Nucleus; Chemicals; Child; Complex; Computer Simulation; Computer software; Data; Deacetylase; Deacetylation; Development; Diabetes Mellitus; Disease; Energy Metabolism; Energy-Generating Resources; Enzymes; Event; Exposure to; Family member; Fasting; Fatty Acids; Functional disorder; Future; Genetic; Glutamic Acid; Health; Heart; Hereditary Disease; Histones; Human; In Vitro; Inborn Errors of Metabolism; Ionic Strengths; K-Series Research Career Programs; Knockout Mice; Lead; Life; Link; Liver; Long-Chain-Acyl-CoA Dehydrogenase; Lysine; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane Proteins; Metabolic; Metabolism; Methods; Mitochondria; Mitochondrial Proteins; Modification; Molecular; Molecular Models; Myocardium; Names; Nerve Degeneration; Obesity; Organ; Palmitates; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Production; Protein Family; Proteins; Publishing; Reaction; Recombinants; Regulation; Research; Site; Site-Directed Mutagenesis; Stress; Structure; Surface; Surveys; Tail; Temperature; Testing; Time; Tissues; Variant; Work; acyl-CoA dehydrogenase; base; chemical reaction; clinically relevant; deprotonation; enzyme deficiency; fatty acid metabolism; fatty acid oxidation; human disease; in vivo; inhibitor/antagonist; member; molecular modeling; mortality; novel; oxidation; oxidative damage; prevent; programs; public health relevance; repaired; research study; transcription factor

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.

描述（由申请人提供）： 申请人研究计划的长期目标是了解负责控制线粒体脂肪酸代谢的调节机制。提出了一个为期两年的项目，以研究酰基-COA脱氢酶（ACAD）蛋白质家族成员赖氨酸残基的乙酰化。 Acads是线粒体黄酮酶，催化脂肪酸氧化的第一步。这些酶的遗传缺陷是最常见的新陈代谢错误之一。另外，肥胖，糖尿病和其他常见疾病与线粒体脂肪酸氧化有关。因为它们在脂肪酸氧化周期中催化限速步骤，所以调节ACAD的因素调节脂肪酸的能量产生速率。初步数据表明，赖氨酸乙酰化的翻译后修饰可以调节长链酰基-COA脱氢酶（LCAD）的活性，两倍。 LCAD和其他线粒体蛋白化为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室中，但在线粒体中却没有鉴定出来。初步数据表明，几种ACAD酶的乙酰化可以化学发生。以浓度大于0.5 mm的乙酰辅酶A纯化的重组学院的孵育导致赖氨酸乙酰化。由于线粒体乙酰-COA浓度可能达到2 mm或更高，因此假设化学乙酰化是为了造成高度氧化组织（如肝脏，肌肉，心脏和棕色脂肪）在高度氧化组织的线粒体中看到的赖氨酸乙酰化现象。提出了两个目标来研究这种现象。目的1是表征Acad酶的非酶赖氨酸乙酰化。这包括对反应条件，乙酰转移酶抑制剂的使用以及多种检测和量化赖氨酸乙酰化的方法的全面调查。目标2是确定ACAD蛋白表面的微环境如何影响赖氨酸乙酰化。基于三维分子建模和其他初步数据，据推测，位于酸性氨基酸附近的赖氨酸尤其容易因酸性残基的主动去质子化而容易修饰。通过与乙酰辅酶A反应的重组学院将进行质谱法，以鉴定乙酰化位点。然后，这些位点将使用分子建模软件和先前发布的ACAD晶体结构在三维空间中进行映射。最后，将使用位置定向的诱变来确定相邻氨基酸对每种鉴定赖氨酸的乙酰化的贡献。拟议的R03项目将与申请人当前的K01职业发展奖协同作用，从而更快地过渡到独立性和更具竞争力的未来R01申请。 公共卫生相关性：线粒体能量代谢的公共卫生相关性变化是许多主要疾病从癌症到糖尿病的主要疾病，并且也被认为是衰老过程的核心。拟议的研究旨在阐明对称为赖氨酸乙酰化的代谢酶修饰的分子事件。了解线粒体酶的赖氨酸乙酰化可能会导致新药的发展以控制代谢，从而预防疾病甚至减慢衰老。