THE ROLE OF ACETYLATION IN MITOCHONDRIAL FUNCTION

乙酰化在线粒体功能中的作用

• 批准号: 8696819
• 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/8696819
• 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; 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.

描述（由申请人提供）：