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使用的大部分能量，是细胞功能所必需的。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。由TCA循环和脂肪酸代谢产生的还原当量被NADH-泛醌还原酶(复合体I)、琥珀酸-泛醌还原酶(复合体II)和ETF-泛醌还原酶用于将膜结合的泛醌还原为泛喹酚。然后泛喹酚被Bc1络合物(络合物III)氧化，电子通过络合物IV(细胞色素c氧化酶)转移到氧中产生水。在这个电子转移过程中，质子被泵过线粒体膜，产生一个质子电化学梯度，被复合体V(三磷酸腺苷合成酶)用来产生三磷酸腺苷。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。代谢蛋白质和大型蛋白质复合体，如线粒体中发现的，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，而基团的去除依赖于几个家族的脱乙酰酶。其中一个家族是NAD依赖的脱乙酰酶家族，称为sirtuins。SIRT3是这个家族的成员，似乎是线粒体内的一种全局脱乙酰酶。在这个项目中，一个SIRT3/-基因敲除的小鼠模型将被用来研究乙酰化在线粒体生理控制中的作用。将使用线粒体模型系统来研究三个特定的目标。第一，线粒体蛋白乙酰化是否影响呼吸链活性和复合体I的活性/失活转变；第二，乙酰化是否影响非常大的1-酮戊二酸/丙酮酸脱氢酶复合体的E3(硫酰胺脱氢酶)组分形成ROS(活性氧物种)。第三，由于线粒体被认为参与了细胞的应激反应，将进行研究，以研究乙酰化如何在心脏缺血/再灌注损伤模型中影响反应。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很好的模型系统。因此，从对照组和SIRT3-/-KO小鼠模型中分离出完整的线粒体、甲氧西林可渗透的线粒体和亚线粒体颗粒，并对其进行呼吸功能检测。研究的重点是呼吸链的化合物I和II，它们控制还原当量进入呼吸链和硫酰胺脱氢酶。研究表明，乙酰化既影响了这些复合体的活性，也影响了它们与其他线粒体蛋白的相互作用，从而影响了线粒体的整体效率，并可能有助于ROS的形成。作为减少呼吸链复合体中等效物的切入点，I是线粒体功能的重要调节因子。众所周知，这种酶经历了一个活性/失活的转变。将进行研究，以确定复合体I的活性/去活性转换是否受乙酰化的影响，以及这是否调节线粒体的功能。分光光度和呼吸测定法用于评估催化活性，蛋白质印迹和一维和二维凝胶电泳法将用于评估蛋白质-蛋白质相互作用。利用SIRT3 KO小鼠模型，将确定心脏组织的属性是否因蛋白质的乙酰化/去乙酰化而改变，如前后调节。 公共卫生相关性： 线粒体是细胞的能量源泉。因此，许多代谢性和退行性疾病被证明改变了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含有线粒体成分。线粒体中的蛋白质修饰可以改变酶的功能。一种类型的改变是蛋白质中发现的氨基酸赖氨酸的乙酰化。Sirtuins是一个从修饰的赖氨酸残基中去除乙酰基的酶家族，线粒体sirtuin SIRT3活性的增加与长寿有关。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。获得的信息将有助于设计治疗困扰退伍军人的疾病的方法。

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.