An epsilon PKC interacting protein in preconditioning

描述（由申请人提供）：epsilon蛋白激酶C （epsilonPKC）同工酶已被明确确定为心脏预适应（PC）的关键介质。因此，研究epsilonpkc选择性信号的分子机制，以更好地了解它如何介导保护，并确定开发epsilonpkc选择性治疗性PC调节剂的潜在途径是非常有意义的。我们目前的研究表明，在心肌细胞颗粒细胞部分中发现的约18 kDa蛋白的体外磷酸化可以作为epsilonPKC激活和epsilonPKC介导的PC的标记物。肽质谱和免疫沉淀分析确定该蛋白是细胞色素c氧化酶（COIV）的IV亚基。此外，该蛋白可能是新生儿心肌细胞（ncm）中epsilonPKC的体内底物。我们假设COIV在心脏缺血保护中起着关键且以前未被描述的作用。先前我们证明了3nm 4- β PMA处理优先激活ncm中的epsilonPKC同工酶。在这个提议中，我们将确定epsilonPKC是否可以调节细胞色素c的活性，并将这些发现与心脏PC联系起来。我们将从经过3 nM 4- β PMA处理和缺氧PC的NMCs中分离线粒体和亚线粒体部分，并监测细胞色素c氧化酶活性，epsilonPKC与COIV的结合或磷酸化，共聚焦和电子显微镜COIV和epsilonPKC共定位研究以及质谱，Cy染料和Pro-Q Diamond方法来鉴定磷酸化蛋白。类似的分析将在从缺血PC的成年大鼠心脏中分离的线粒体和线粒体组分上进行。该提案的具体目的是：目的1：确定epsilonPKC对新生儿心肌细胞细胞色素c氧化酶的影响；目的二：探讨epsilonPKC对细胞色素c氧化酶在新生儿心肌细胞预处理中的调节作用。目的三：探讨epsilonPKC对缺血/再灌注和缺血预处理大鼠心肌细胞色素c氧化酶的调节作用。我们的工作将集中在由epsilonPKC同工酶调节的PC范式中的一个新的机制事件。更好地了解与PC相关的epsilonPKC分子和细胞靶点，将为心肌梗死风险患者的心脏保护策略的治疗应用提供机会。

DESCRIPTION (provided by applicant): The epsilon protein kinase C (epsilonPKC) isozyme has been clearly established as a key mediator of cardiac preconditioning (PC). It is therefore of great interest to study the molecular mechanisms of epsilonPKC-selective signaling to better understand how it mediates protection and to identify potential avenues for the development of epsilonPKC-selective therapeutic modulators of PC. Our current studies indicate that in vitro phosphorylation of an approximately 18 kDa protein found in the particulate cell fraction of cardiac myocytes can be used as a marker of epsilonPKC activation and epsilonPKC-mediated PC. Peptide mass spectrometry and immunoprecipitation analyses have determined that this protein is the IV subunit of cytochrome c oxidase (COIV). Further, this protein is likely an in vivo substrate of epsilonPKC in neonatal cardiac myocytes (NCMs). We hypothesize that COIV plays a key and previously uncharacterized role in cardiac protection against ischemia. Previously we demonstrated that 3 nM 4-beta PMA treatment preferentially activates the epsilonPKC isozyme in NCMs. In this proposal we will determine if epsilonPKC can regulate cytochrome c activity and relate these findings to cardiac PC. We will isolate mitochondria and submitochondrial fractions from NMCs subjected to 3 nM 4-beta PMA treatment and hypoxic PC and monitor cytochrome c oxidase activity, epsilonPKC binding to or phosphorylation of COIV, confocal and electron microscopy COIV and epsilonPKC co-localization studies and mass spectrometric, Cy dye and Pro-Q Diamond methodologies to identify phospho-proteins. Similar analyses will be performed on mitochondria and mitochondrial fractions isolated from adult rat hearts subjected to ischemic PC. The specific aims of this proposal will be: Aim i: To determine the effects of epsilonPKC on cytochrome c oxidase in neonatal cardiac myocytes.; Aim ii To determine the role of epsilonPKC modulation of cytochrome c oxidase in neonatal cardiac myocyte preconditioning and Aim iii: To determine the role of epsilonPKC in the modulation of cytochrome c oxidase in adult rat myocardium exposed to ischemia/reperfusion and ischemic preconditioning. Our work will focus on a novel mechanistic event in the PC paradigm modulated by the epsilonPKC isozyme. A better understanding of the molecular and cellular epsilonPKC targets involved in PC will improve opportunities for the therapeutic application of cardioprotective strategies for patients at risk for myocardial infarction.