Modulation of Oxidative phosphorylation by mitochondrial soluble adenylyl cyclase

描述(申请人提供)：线粒体蛋白的磷酸化是调节氧化磷酸化(OXPHOS)和维持能量平衡的一种快速而有效的方法。线粒体PKA通过蛋白磷酸化来调节电子传递链上的酶，蛋白磷酸化受一条信号通路的调节，涉及线粒体可溶性腺苷环化酶(SAC)，产生cAMP激活PKA。这一信号级联起到代谢传感器的作用，调节线粒体的能量转换。细胞色素氧化酶(COX)是ETC通量的起搏器，其活性受其亚基的磷酸化和ATP变构抑制的调节。我们证明COX是信号通路的靶标，并确定COX IV亚基(COXIV)是磷酸化的靶标。我们还证明了COXIV-1中S58的磷酸化是COX活性的调节因素。我们认为，这一调节途径参与了对OXPHOS缺陷的代谢适应性反应，并可能成为药物干预的靶点。本项目的目的是了解线粒体内SAC-cAMP-PKA信号和s58 COXIV-1磷酸化在体内的生理意义。我们将了解这个系统在健康组织和受线粒体缺陷影响的组织中是如何运作的。为此，我们将产生和研究小鼠，在其中的调控途径的分子成员是基因修饰的，并评估结果的临床表型和线粒体生化。在目标1中，我们将获得表达选择性靶向线粒体基质的可诱导SAC(mito-SAC)的转基因小鼠，这有望产生高基础代谢。在AIM2中，我们将产生COXIV-1 S58A敲门小鼠，由于缺乏磷酸化位点，因此无法上调ETC通量和增强ATP的产生，导致运动耐量降低，葡萄糖利用减少，产热受损，脂肪储存增加。在目标3中，我们将研究SAC-cAMP-PKA在COX条件性和可诱导性遗传中断引起的OXPHOS缺陷小鼠模型中的调节。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的调节和蛋白磷酸化是如何进行的，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.