Physiological Modulators of Cytochrome Oxidase Function

描述（由申请人提供）：细胞色素c氧化酶（CytOX）是线粒体呼吸链的末端氧化酶。细胞x结构和活性的改变与多种退行性疾病和心肌损伤有关。初步结果表明，通过PKA介导的亚基IV、Vb和亚基i的磷酸化，以及在较小程度上抑制PKA介导的缺氧和缺血/再灌注中CytOX的活性。一个令人兴奋的观察结果是，用PKA特异性抑制剂H89预处理细胞和心脏，可以显著逆转缺氧介导的细胞变化和缺血介导的心肌损伤。在此基础上，更新应用的重点是验证cAMP介导的CytOX过度磷酸化的假设，即由于ROS的过量产生而改变酶的活性并对细胞/组织产生有害影响。建议采用生物化学、细胞生物学和转基因相结合的方法来验证假设，具体方法如下：1)。小鼠巨噬细胞细胞系在缺氧条件下的细胞x和兔心脏在实验性缺血/再灌注条件下的细胞x，将在蛋白脂质体系统中进行动力学参数（Km和TN）、质子泵送和ROS产生的表征，并研究PKA抑制剂在逆转这些变化中的作用。I、IV和Vb亚基的磷酸化位点将通过指纹分析和串联质谱分析相结合的方法进行定位。2）. 我们将研究巨噬细胞中有条件地去除CytOX IV和Vb亚基以及磷酸化位点突变亚基替代对缺氧诱导损伤的影响。3)。最后，通过产生磷酸化位点突变的CytOX IV和Vb基因小鼠系，进一步研究核亚基在CytOX组装和功能中的作用。总体目标是确定H89介导的心肌缺血损伤保护的分子基础。

DESCRIPTION (provided by applicant): Cytochrome c oxidase (CytOX) is the terminal oxidase of the mitochondrial respiratory chain. Altered CytOX structure and activity are associated with a wide spectrum of degenerative diseases, and myocardial injury. Preliminary results show that CytOX activity in hypoxia and ischemia/reperfusion is markedly inhibited through PKA mediated phosphorylation of subunits IV, and Vb and to a lesser extent subunit I. An exciting observation is that hypoxia mediated changes in cells and ischemia mediated myocardial injury can be substantially reversed by pre-treating cells, and or, heart with PKA specific inhibitor, H89. Based on this, the renewal application is focused to test the hypothesis that cAMP mediated hyperphosphorylation of CytOX alters the enzyme activity and exerts deleterious effects on cells/tissues due to overproduction of ROS. It is proposed to use a combination of biochemical, cell biological, and transgenic approaches to test the hypothesis as follows: 1). CytOX from murine macrophage cell line subjected to hypoxia, and rabbit heart subjected to experimental ischemia/reperfusion, will be characterized with respect to kinetic parameters (Km and TN), proton pumping, and ROS production in a proteoliposome system, and the effects of PKA inhibitors in reversing these changes will be studied. The sites of phosphorylation of subunits I, IV and Vb will be mapped by a combination of fingerprint analysis and tandem MS-MS analysis of peptides. 2). The effects of conditional depletion of CytOX IV and Vb subunits and replacement with phosphorylation site mutated subunits in macrophage cells on hypoxia induced injury will be studied. 3). Finally, the role of nuclear subunits in CytOX assembly and function will be further studied by generating mouse lines with phosphorylation site mutated CytOX IV and Vb genes. The overall goal is to determine the molecular basis of H89 mediated protection against ischemic injury to the myocardium.