THE ROLE OF ACETYLATION IN MITOCHONDRIAL FUNCTION

乙酰化在线粒体功能中的作用

• 批准号: 8254308
• 负责人: Gary Cecchini
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8254308
• 关键词: Acetyl Coenzyme A; Acetylation; Acetyltransferase; Address; Affect; Aging; Alamethicin; Amino Acids; Ammonium; Animals; Attention; Binding; Biological Assay; Biological Models; Cardiac; Cell Survival; Cell physiology; Cells; Citric Acid Cycle; Complex; Deacetylase; Deacetylation; Degenerative Disorder; Diabetes Mellitus; Disease; Electron Transport; Electron Transport Complex III; Energy-Generating Resources; Enzymes; Excision; Family; Family member; Generations; Heart; Heart Diseases; Infarction; Investigation; Ions; Ketoglutarate Dehydrogenase Complex; Knock-out; Knockout Mice; Light; Liver; Longevity; Lysine; Malignant Neoplasms; Membrane; Metabolic; Metabolic Diseases; Metabolic stress; Metabolism; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Multienzyme Complexes; Mus; NADH dehydrogenase (ubiquinone); Nerve Degeneration; Neurodegenerative Disorders; Oxidoreductase; Oxygen; Physiology; Play; Post-Translational Protein Processing; Process; Property; Protein Acetylation; Proteins; Proton Pump; Protons; Pyruvate Dehydrogenase Complex; Quinone Reductases; Reaction; Reactive Oxygen Species; Recovery; Reperfusion Injury; Respiratory Chain; Respiratory physiology; Role; Sir2-like Deacetylases; Sirtuins; Submitochondrial Particles; Succinate dehydrogenase (ubiquinone); Tissues; Two-Dimensional Gel Electrophoresis; Ubiquinone; Veterans; Water; Western Blotting; amino group; biological adaptation to stress; body system; cell growth regulation; conditioning; conformational conversion; cytochrome c oxidase; design; dihydrolipoamide dehydrogenase; fatty acid metabolism; hemodynamics; mitochondrial dysfunction; mitochondrial membrane; mouse model; oligomycin sensitivity-conferring protein; patient population; protein complex; protein protein interaction; public health relevance; respiratory; response; therapy design; ubiquinol

DESCRIPTION (provided by applicant): Mitochondria generate much of the energy used by animal cells and are essential for cellular function. This energy generating apparatus involves the interplay between the tricarboxylic acid (TCA) cycle and the membrane-bound electron transport chain. Reducing equivalents generated by the TCA cycle and fatty acid metabolism are used by NADH-ubiquinone reductase (complex I), succinate-ubiquinone reductase (complex II), and ETF-quinone reductase to reduce membrane- bound ubiquinone to ubiquinol. Ubiquinol is then oxidized by the bc1 complex (complex III) and electrons transferred to oxygen through complex IV (cytochrome c oxidase) to produce water. During this electron transfer process protons are pumped across the mitochondrial membrane generating a proton electrochemical gradient used by complex V (ATP synthase) to generate ATP. Numerous studies show that posttranslational modification of proteins can regulate their function. Reversible acetylation of lysine residues is one such modification that is receiving increasing attention. Metabolic proteins and large protein complexes, such as found in the mitochondrion, are particularly prone to acetylation/deacetylation reactions. Lysine acetylation relies on acetyl coenzyme A (AcCoA) as the acetyl donor whereas removal of the group relies on several families of deacetylases. One such family is the NAD+dependent family of deacetylases referred to as sirtuins. SIRT3 is a member of this family and appears to be a global deacetylase within the mitochondrion. In this project a SIRT3-/- knockout mouse model will be used to investigate the role of acetylation in control of mitochondrial physiology. Three specific aims will be investigated using mitochondrial model systems. First, does mitochondrial protein acetylation affect respiratory chain activity and the active/deactive transition of complex I. Second, does acetylation effect ROS (reactive oxygen species) formation from the E3 (lipoamide dehydrogenase) component of the very large 1-ketoglutarte/pyruvate dehydrogenase complexes. Third, as mitochondria are suggested to be involved in the stress response of cells, studies will be undertaken to investigate how acetylation affects the response in a cardiac ischemia/reperfusion injury model. The focus of the studies will be on heart and liver tissue since both of these provide excellent model systems for investigation of mitochondrial function. Therefore, tissues from control and SIRT3-/- KO mouse models will be isolated and intact mitochondria, alamethicin permeabilized mitochondria, and submitochondrial particles prepared and assayed for respiratory function. A focus of the studies is on complexes I and II of the respiratory chain which control the entry of reducing equivalents into the respiratory chain and on lipoamide dehydrogenase. It is suggested that acetylation affects both the activity of these complexes and their interaction with other mitochondrial proteins which affects overall mitochondrial efficiency and potentially contributes to ROS formation. As the entry point for reducing equivalents into the respiratory chain complex I is an important regulator of mitochondrial function. It is known that this enzyme undergoes an active/deactive transition. Studies will be done to determine if the active/deactive transition of complex I is affected by acetylation and if this modulates mitochondrial function. Spectrophotometric and respirometry methods are used to assess catalytic activity, and Western blots and 1D- and 2D-gel electrophoresis will be used to assess protein-protein interactions. Using the SIRT3 KO mouse model, it will be determined if the properties of cardiac tissue such as pre- and post-conditioning are altered by acetylation/deacetylation of proteins. PUBLIC HEALTH RELEVANCE: Mitochondria are the energy powerhouse of the cell. Therefore it is not surprising that many metabolic and degenerative diseases have been shown to have altered mitochondrial function. In the VA patient population diseases such as diabetes, heart disease, aging, and neurodegenerative disease have been shown to have a mitochondrial component. Protein modifications in mitochondria can alter enzyme function. One type of alteration is acetylation of the amino acid lysine found in proteins. Sirtuins are a family of enzymes that remove acetyl groups from modified lysine residues and increased activity of the mitochondrial sirtuin SIRT3 is associated with longevity. The studies described in this application are designed to shed light on the role of acetylation in controlling mitochondrial function during periods of metabolic stress and how this contributes to disease. The information obtained will aid in design of treatments for diseases that afflict veterans.

描述（由申请人提供）： 线粒体产生动物细胞使用的大部分能量，对于细胞功能至关重要。这种能量产生装置涉及三羧酸（TCA）循环和膜结合电子传输链之间的相互作用。 TCA 循环和脂肪酸代谢产生的还原当量被 NADH-泛醌还原酶（复合物 I）、琥珀酸-泛醌还原酶（复合物 II）和 ETF-醌还原酶用来将膜结合的泛醌还原为泛醇。然后泛醇被 bc1 复合物（复合物 III）氧化，电子通过复合物 IV（细胞色素 c 氧化酶）转移到氧气，产生水。在此电子转移过程中，质子被泵送穿过线粒体膜，产生质子电化学梯度，复合物 V（ATP 合酶）使用该梯度来生成 ATP。 大量研究表明蛋白质的翻译后修饰可以调节其功能。赖氨酸残基的可逆乙酰化就是一种受到越来越多关注的修饰。代谢蛋白质和大型蛋白质复合物（例如在线粒体中发现的）特别容易发生乙酰化/脱乙酰化反应。赖氨酸乙酰化依赖于乙酰辅酶 A (AcCoA) 作为乙酰基供体，而该基团的去除则依赖于几个脱乙酰酶家族。其中一个家族是NAD+依赖性脱乙酰酶家族，称为sirtuins。 SIRT3 是该家族的成员，似乎是线粒体内的全局脱乙酰酶。在该项目中，SIRT3-/- 敲除小鼠模型将用于研究乙酰化在控制线粒体生理学中的作用。 将使用线粒体模型系统研究三个具体目标。首先，线粒体蛋白乙酰化是否会影响呼吸链活性以及复合物 I 的活性/失活转变。其次，乙酰化是否会影响非常大的 1-酮戊二酸/丙酮酸脱氢酶的 E3（硫辛酰胺脱氢酶）成分形成 ROS（活性氧）复合物。第三，由于线粒体被认为参与细胞的应激反应，因此将进行研究以调查乙酰化如何影响心脏缺血/再灌注损伤模型中的反应。研究的重点将是心脏和肝脏组织，因为这两者都为线粒体功能的研究提供了出色的模型系统。因此，将分离来自对照和SIRT3-/-KO小鼠模型的组织，并制备完整的线粒体、阿拉甲辛透化的线粒体和亚软骨颗粒，并测定其呼吸功能。研究的重点是控制还原当量进入呼吸链的呼吸链复合物 I 和 II 以及硫辛酰胺脱氢酶。研究表明乙酰化会影响这些复合物的活性以及它们与其他线粒体蛋白的相互作用，从而影响线粒体的整体效率并可能有助于 ROS 的形成。作为呼吸链复合物 I 还原当量的入口点，它是线粒体功能的重要调节因子。众所周知，这种酶经历活性/失活转变。将进行研究以确定复合物 I 的活性/失活转变是否受到乙酰化的影响以及这是否调节线粒体功能。分光光度法和呼吸测定法用于评估催化活性，蛋白质印迹以及一维和二维凝胶电泳将用于评估蛋白质-蛋白质相互作用。使用 SIRT3 KO 小鼠模型，将确定心脏组织的特性（例如预处理和后处理）是否会因蛋白质的乙酰化/脱乙酰化而改变。 公共卫生相关性： 线粒体是细胞的能量发源地。因此，许多代谢和退行性疾病已被证明会改变线粒体功能也就不足为奇了。在 VA 患者群体中，糖尿病、心脏病、衰老和神经退行性疾病等疾病已被证明与线粒体成分有关。线粒体中的蛋白质修饰可以改变酶的功能。一种类型的改变是蛋白质中氨基酸赖氨酸的乙酰化。 Sirtuins 是一类酶，可从修饰的赖氨酸残基中去除乙酰基，线粒体 Sirtuin SIRT3 活性的增加与长寿相关。本申请中描述的研究旨在阐明乙酰化在代谢应激期间控制线粒体功能的作用以及它如何导致疾病。获得的信息将有助于设计治疗退伍军人疾病的方法。