Physiological Modulators of Cytochrome Oxidase Function

超出提供的空间。细胞色素c氧化酶(CytOX)是线粒体呼吸链的末端氧化酶。细胞氧合酶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，在低氧和缺血/再灌流中，CytOX活性通过PKA介导的亚基IV、Vb和I亚基的磷酸化而受到显著抑制。令人兴奋的观察是，用PKA特异性抑制剂H89预处理细胞、土地或心脏，可显著逆转缺氧介导的I细胞变化和缺血介导的心肌损伤。在此基础上，更新应用重点是验证cAMP介导的CytOX过度磷酸化改变酶活性的假说，该假说由于ROS的过量产生而对细胞/组织产生有害影响。建议使用生化、细胞生物学和转基因方法的组合来检验这一假设，如下所示：1)。从低氧小鼠巨噬细胞系和实验性缺血/再灌流的兔心脏J获得的CytOX，将在蛋白脂质体系统中从动力学参数(Km和Tn)、质子泵和ROS产生方面进行表征，并将研究PKA抑制剂在逆转这些变化中的作用。亚基I、IV和Vb的磷酸化位点将通过多肽的指纹分析和串联MS-MS分析相结合来绘制。2)。我们将研究巨噬细胞中CytOX IV和VB亚基的条件性缺失和磷酸化位点突变亚基的替代对缺氧损伤的影响。3)。最后，通过建立具有磷酸化位点突变的CytOX IV和VB基因的小鼠株系，进一步研究核亚单位在CytOX组装和功能中的作用。总的目标是确定H89介导的抗心肌缺血损伤的分子基础。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 Icells and ischemia mediated myocardial injury can be substantially reversed by pre-treating cells, land or, heart with PKA specific inhibitor, H89. Based on this, the renewal application is focused to Itest the hypothesis that cAMP mediated hyperphosphorylation of CytOX alters the enzyme activity land exerts deleterious effects on cells/tissues due to overproduction of ROS. It is proposed to use a I combination of biochemical, cell biological, and transgenic approaches to test the hypothesis as Ifollows: 1). CytOX from murine macrophage cell line subjected to hypoxia, and rabbit heart Jsubjected 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. PERFORMANCE SITE ========================================Section End===========================================