Modulation of Oxidative phosphorylation by mitochondrial soluble adenylyl cyclase

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方式。线粒体PKA通过蛋白质磷酸化调节电子传递链的酶，其由涉及线粒体可溶性腺苷酸环化酶（sAC）的信号传导途径调节，产生激活PKA的cAMP。这种信号级联作为一种代谢传感器，调节线粒体中的能量转换。细胞色素氧化酶（考克斯）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。我们发现，考克斯是一个目标的信号转导途径，并确定了亚基IV的考克斯（COXIV）作为磷酸化的目标。我们还证明了COXIV-1中S58的磷酸化是调节考克斯活性的原因。我们建议，这一调节途径参与代谢适应性反应OXPHOS缺陷，并可能成为药物干预的目标。本项目的目标是了解线粒体内sAC-cAMP-PKA信号传导和S58 COXIV-1磷酸化在体内的生理意义。我们将了解该系统如何在健康组织和受线粒体缺陷影响的组织中发挥作用。为此，我们将产生和研究小鼠，其中调节途径的分子参与者被遗传修饰，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的诱导型sAC（mito-sAC）的转基因小鼠，预期其产生高基础代谢。在aim2中，我们将产生COXIV-1 S58A敲入小鼠，缺乏磷酸化位点，因此不能上调ETC通量和增强ATP产生，导致运动不耐受，葡萄糖利用减少，产热受损和脂肪储存增加。在目的3中，我们将研究由条件性和诱导性考克斯遗传破坏引起的OXPHOS缺陷小鼠模型中的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sAC-cAMP-PKA调节和蛋白磷酸化在受影响组织的线粒体中的作用，并将其与疾病进展相关联。

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.