THE ROLE OF ACETYLATION IN MITOCHONDRIAL FUNCTION

描述（由申请人提供）： 线粒体产生动物细胞所需的大部分能量，对细胞功能至关重要。这种能量产生装置涉及三羧酸（TCA）循环和膜结合电子传递链之间的相互作用。由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）、琥珀酸酯-泛醌还原酶（复合物II）和ETF-醌还原酶用于将膜结合的泛醌还原成泛醇。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）转移到氧气中，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，由复合物V（ATP合酶）用于产生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是一种受到越来越多关注的修饰。代谢蛋白质和大蛋白质复合物，如在胰蛋白酶中发现的，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）作为乙酰供体，而基团的去除依赖于几个脱乙酰酶家族。一个这样的家族是NAD+依赖性的脱乙酰酶家族，称为沉默调节蛋白。SIRT3是这个家族的成员，似乎是一个全球性的脱乙酰基酶内的蛋白质。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。 三个具体的目标将使用线粒体模型系统进行调查。首先，线粒体蛋白乙酰化是否影响呼吸链活性和复合物I的活性/失活转变。第二，乙酰化是否影响ROS（活性氧）从E3（硫辛酰胺脱氢酶）组分的非常大的1-酮戊二酸/丙酮酸脱氢酶复合物的形成。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。研究的重点将放在心脏和肝脏组织上，因为这两种组织都为研究线粒体功能提供了极好的模型系统。因此，将分离对照和SIRT3-/-KO小鼠模型的组织，并制备完整线粒体、丙甲霉素透化线粒体和亚线粒体颗粒，并测定其呼吸功能。研究的重点是呼吸链的复合物I和II，其控制还原当量进入呼吸链和硫辛酰胺脱氢酶。这表明，乙酰化影响这些复合物的活性和它们与其他线粒体蛋白的相互作用，从而影响整体线粒体效率，并可能有助于ROS的形成。作为还原当量进入呼吸链的入口点，复合物I是线粒体功能的重要调节剂。已知这种酶经历活性/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，蛋白质印迹和1D-和2D-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，将确定心脏组织的性质（例如预处理和后处理）是否通过蛋白质的乙酰化/脱乙酰化而改变。 公共卫生相关性： 线粒体是细胞的能量发电站。因此，许多代谢和退行性疾病已被证明具有改变的线粒体功能并不奇怪。在VA患者人群中，已显示糖尿病、心脏病、衰老和神经退行性疾病等疾病具有线粒体组分。线粒体中的蛋白质修饰可以改变酶的功能。一种类型的改变是在蛋白质中发现的氨基酸赖氨酸的乙酰化。沉默调节蛋白是从修饰的赖氨酸残基去除乙酰基的酶家族，并且线粒体沉默调节蛋白SIRT3的活性增加与寿命相关。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这如何导致疾病。所获得的信息将有助于设计折磨退伍军人的疾病的治疗方法。

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.