Role of Protein Malonylation in Regulating Mitochondrial Function

蛋白质丙二酰化在调节线粒体功能中的作用

• 批准号: 9049922
• 负责人: Kelsey Fisher-Wellman
• 金额: $ 5.42万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9049922
• 关键词: ATP Synthesis Pathway; Ablation; Acetylation; Acyl Coenzyme A; Acylation; Aging; Burn injury; Caloric Restriction; Chronic; Citrates; Citric Acid Cycle; Computer Simulation; Coupled; Data; Data Set; Development; Diet; Disease; Employee Strikes; Energy Metabolism; Enzymes; Event; Exercise; Failure; Fatty acid glycerol esters; Fiber; Functional disorder; Genetic; Heart Diseases; Homeostasis; Hydrogen Peroxide; Isocitrate Dehydrogenase; Kinetics; Knock-out; Knockout Mice; Link; Liver; Lysine; Malonyl Coenzyme A; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Mus; Muscle; National Research Service Awards; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Obesity; Organ; Oxidation-Reduction; Oxidoreductase; Peptides; Phenotype; Physiology; Post-Translational Protein Processing; Predisposition; Proteins; Public Health; Pyruvate Dehydrogenase Complex; Reporting; Respiratory physiology; Role; Site; Skeletal Muscle; Testing; Tissues; Western Blotting; enzyme activity; fatty acid oxidation; feeding; follow-up; genetic approach; malonyl-CoA decarboxylase; mitochondrial dysfunction; mouse model; novel; novel therapeutics; oxidation; prevent; public health relevance; research study; respiratory

 DESCRIPTION (provided by applicant): Lysine acylation has emerged as a prominent post-translational modification (PTM) within mitochondrial proteins. Increased susceptibility of mitochondrial proteins to acylation is a function of the alkaline pH of the matrix, in conjunction with the relatively high concentration of acyl-CoA intermediates. Protein acylation varies as a function of the matrix acyl-CoA pool and is counterbalanced by the activity of various mitochondrial- localized deacylases (SIRT3, 4, 5). As such, metabolic conditions know to increase free acyl-CoAs (e.g., high- fat diet, caloric restriction), as well as genetic knockout of either SIRT3 or SIRT5 have revealed a wide range of acetyl and acyl (e.g., malonylation, succinylation, glutarylation) modifications throughout various mitochondrial proteins. Although in some cases lysine acylation has been linked to altered enzymatic flux (e.g., lysine acetylation inhibits is citrate dehydrogenase), the functional relevance of these modifications in the context of mitochondrial physiology and disease is largely unknown. Malonyl-CoA decarboxylase (MCD) is an enzyme responsible for the degradation of malonyl-CoA, and genetic ablation of the enzyme in MCD-/- mice results in elevated tissue levels of malonyl-CoA. Our preliminary studies show that MCD in muscle and liver is predominantly localized to the mitochondria. Moreover, Western blot assessment of global malonylation in MCD-/- mice revealed striking elevations in malonylated proteins, particularly in the mitochondrial compartment. Follow up experiments using LC mass spectrometry (MS)/MS revealed widespread hypermalonylation in skeletal muscle tissue of muscle-specific MCD deficient mice (MCDMCK-/-). Included among identified targets of malonylation were two of the three subunits of the pyruvate dehydrogenase complex (PDC), as well as enzymes involved in beta-oxidation, ATP synthesis, the TCA cycle and redox homeostasis. Thus, the current NRSA application will test the overarching hypothesis that MCD defends mitochondrial function in muscle and liver by protecting against hypermalonylation of matrix proteins. In Aim 1 we will identify malonylated lysine residues in both skeletal muscle and liver of tissue specific MCD deficient mice under low fat and high fat fed conditions and then compare these sites with those previously reported for SIRT5-/- mice. In Aim 2 we will focus on hits identified in preliminary studies and SA1 to determine if the hypermalonylation phenotype of the MCD deficient mice is accompanied by alternations in mitochondrial function (e.g., enzyme activities, respiratory kinetics and H2O2 emission/redox homeostasis). Lastly, in Aim 3 we will use a variety of pharmacological and genetic approaches to alter protein malonylation, coupled with targeted LC-MS(MS), to test the hypothesis that protein malonylation exists as a novel regulator of the PDC. Results could implicate lysine acylation as a cause of mitochondrial failure in the context of aging and/or chronic metabolic disorders.

 描述（通过应用程序提供）：赖氨酸酰基-COA池，并与各种线粒体局部脱酰酶的活性相抵消（SIRT3，4，5）。因此，代谢疾病知道会增加游离酰基-COA（例如，高脂饮食，热量限制），以及遗传敲除 SIRT3或SIRT5都揭示了各种线粒体蛋白质的各种乙酰基和酰基（例如，丙歌化，琥珀酰化，谷胱甘肽化）的修饰。尽管在某些情况下，赖氨酸乙酰基已与酶通通量改变有关（例如，赖氨酸乙酰化抑制是柠檬酸脱氢酶），但在线粒体生理学和疾病的背景下，这些修饰的功能相关性在很大程度上是未知的。 Malonyl-COA脱羧酶（MCD）是导致丙酰coA降解的酶，MCD - / - 小鼠中该酶的遗传消融会导致丙次型-COA的组织水平升高。我们的初步研究表明，肌肉和肝脏中的MCD主要定位于线粒体。此外，对MCD - / - 小鼠全球误导化的蛋白质印迹评估显示，丙二酰化蛋白，特别是在线粒体区室中的升高。使用LC质谱法（MS）/MS的后续实验显示，肌肉特异性MCD缺乏小鼠的骨骼肌组织中宽度超隆式化（MCDMCK - / - ）。丙歌式化靶标的包括丙酮酸脱氢酶复合物（PDC）的三个亚基中的两个，以及参与β-氧化，ATP合成，TCA循环和氧化还原稳态的酶。这是当前的NRSA应用将检验总体假设，即MCD通过预防基质蛋白的高氨基率来捍卫肌肉和肝脏中的线粒体功能。在AIM 1中，我们将在低脂肪和高脂肪喂食条件下识别骨骼肌和组织特异性MCD缺乏小鼠的误导性歌词，然后将这些位点与先前报道的SIRT5 - / - 小鼠进行比较。在AIM 2中，我们将重点关注初步研究和SA1中鉴定的命中，以确定MCD缺乏小鼠的高氨基率表型是否伴随着线粒体功能的改变（例如，酶活性，呼吸动力学和H2O2发射/H2O2发射/氧化还原稳态）。最后，在AIM 3中，我们将使用各种药物和遗传方法来改变蛋白质误导性，再加上靶向LC-MS（MS），以测试蛋白质误导性作为PDC的新调节剂的假设。结果可能暗示赖氨酸酰化是在衰老和/或慢性代谢疾病的背景下导致线粒体衰竭的原因。