Modulation of Oxidative phosphorylation by mitochondrial soluble adenylyl cyclase

线粒体可溶性腺苷酸环化酶对氧化磷酸化的调节

• 批准号: 8332758
• 负责人: Giovanni Manfredi
• 金额: $ 35.49万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8332758
• 关键词: Adenylate Cyclase; Affect; Amino Acids; Basal metabolic rate; Biochemical; Biochemistry; Carbon Dioxide; Cells; Characteristics; Citric Acid Cycle; Clinical; Consumption; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytochrome-c Oxidase Deficiency; Data; Defect; Disease; Disease Progression; Electron Transport; Enzymes; Excision; Exercise; Fatty acid glycerol esters; Genetic; Glucose; Goals; Health; Heart; Homeostasis; Homozygote; Intervention; Investigation; Knock-in Mouse; Laboratories; Life; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Mitochondrial Diseases; Mitochondrial Matrix; Mitochondrial Proteins; Modeling; Molecular; Mus; Nutrient; Outcome; Oxidative Phosphorylation; Pacemakers; Pathway interactions; Phosphorylation; Physiological; Production; Protein Dephosphorylation; Proteins; Regulation; Regulatory Pathway; Running; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Speed; System; Thermogenesis; Tissues; Transgenic Mice; Work; clinical phenotype; coping; cytochrome c oxidase; human disease; in vivo; mitochondrial dysfunction; mouse model; mutant; oxidation; response; sensor

DESCRIPTION (provided by applicant): Phosphorylation of mitochondrial proteins is a rapid and efficient way to regulate oxidative phosphorylation (OXPHOS) and maintain energy homeostasis. Mitochondrial PKA modulates enzymes of the electron transfer chain through protein phosphorylation, which is regulated by a signaling pathway, involving mitochondrial soluble adenylyl cyclase (sAC), generating cAMP that activates PKA. This signaling cascade serves as a metabolic sensor that modulates energy conversion in mitochondria. Cytochrome oxidase (COX) is a pacemaker of ETC fluxes, whose activity is modulated by phosphorylation of its subunits and by ATP allosteric inhibition. We showed that COX is a target of the signaling pathway and identified subunit IV of COX (COXIV) as a target for phosphorylation. We also demonstrated that phosphorylation of S58 in COXIV- 1 is responsible for modulation of COX activity. We propose that this regulatory pathway participates to metabolic adaptive responses to OXPHOS defects and could be a target for pharmacological intervention. The goal of this project is to understand the physiological implications of intramitochondrial sAC-cAMP- PKA signaling and S58 COXIV-1 phosphorylation, in vivo. We will understand how this system behaves in healthy tissues and in tissues affected by mitochondrial defects. To this end, we will generate and study mice, in which the molecular players of the regulatory pathway are genetically modified and assess the outcomes on clinical phenotype and mitochondrial biochemistry. In aim 1, we will generate transgenic mice expressing inducible sAC selectively targeted to the mitochondrial matrix (mito-sAC), which is expected produce high basal metabolism. In aim2, we will generate COXIV-1 S58A knockin mice, lacking the phosphorylated site, and thus incapable of up-regulating ETC fluxes and enhancing ATP production, resulting in exercise intolerance, decreased glucose utilization, impaired thermogenesis, and increased fat storage. In aim 3, we will investigate sAC-cAMP-PKA modulation in a mouse model of OXPHOS defect caused by conditional and inducible genetic disruption of COX. These mice recapitulate biochemical and clinical characteristics of mitochondrial diseases. We will assess how sAC-cAMP-PKA modulation and protein phosphorylation in mitochondria from affected tissues and correlate it with disease progression.

描述（由申请人提供）：线粒体蛋白的磷酸化是调节氧化磷酸化（OXPHOS）和维持能量稳态的快速有效的方法。线粒体 PKA 通过蛋白质磷酸化调节电子传递链的酶，该磷酸化受信号通路调节，涉及线粒体可溶性腺苷酸环化酶 (sAC)，产生激活 PKA 的 cAMP。这种信号级联充当调节线粒体能量转换的代谢传感器。细胞色素氧化酶 (COX) 是 ETC 通量的起搏器，其活性受其亚基磷酸化和 ATP 变构抑制的调节。我们证明 COX 是信号通路的靶点，并确定 COX 的 IV 亚基 (COXIV) 作为磷酸化的靶点。我们还证明了 COXIV-1 中 S58 的磷酸化负责调节 COX 活性。我们认为该调节途径参与了对 OXPHOS 缺陷的代谢适应性反应，并且可能成为药物干预的目标。该项目的目标是了解体内线粒体内 sAC-cAMP-PKA 信号传导和 S58 COXIV-1 磷酸化的生理学意义。我们将了解该系统在健康组织和受线粒体缺陷影响的组织中的行为方式。为此，我们将生成并研究小鼠，其中调节途径的分子参与者经过基因改造，并评估临床表型和线粒体生物化学的结果。在目标 1 中，我们将产生表达选择性靶向线粒体基质 (mito-sAC) 的可诱导 sAC 的转基因小鼠，预计其会产生高基础代谢。在 Target2 中，我们将生成 COXIV-1 S58A 敲入小鼠，该小鼠缺乏磷酸化位点，因此无法上调 ETC 通量和增强 ATP 产生，从而导致运动不耐受、葡萄糖利用率降低、产热受损和脂肪储存增加。在目标 3 中，我们将在由条件性和诱导性 COX 遗传破坏引起的 OXPHOS 缺陷小鼠模型中研究 sAC-cAMP-PKA 调节。这些小鼠概括了线粒体疾病的生化和临床特征。我们将评估受影响组织线粒体中 sAC-cAMP-PKA 的调节和蛋白质磷酸化的情况，并将其与疾病进展相关联。