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epsilon PKC选择性信号转导的分子机制，以更好地了解它是如何介导保护的，并确定开发epsilon PKC选择性PC治疗调节剂的潜在途径是非常有意义的。我们目前的研究表明，在体外，在心肌细胞颗粒细胞中发现的一个约18 kDa的蛋白的磷酸化可以作为epsilonPKC激活和epsilonPKC介导的PC的标志。多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(NCMS)中epsilonPKC的活体底物。我们假设COIV在抗缺血的心脏保护中扮演着一个关键的、以前未被描述的角色。以前我们证明了3 NM 4-βPMA处理优先激活NCMS中的epsilonPKC同工酶。在这项提案中，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-beta PMA处理和低氧PC的NMC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下的COIV和epsilonPKC的共定位研究以及质谱学、Cy染料和Pro-Q Diamond方法来鉴定磷酸化蛋白。类似的分析也将对线粒体和从成年大鼠心脏中分离出来的线粒体部分进行。本研究的具体目的是：目的一：确定epsilonPKC对新生大鼠心肌细胞细胞色素c氧化酶的影响。目的ii确定epsilonPKC对细胞色素c氧化酶的调节在新生心肌细胞预适应中的作用。目的III：确定epsilonPKC在成年大鼠心肌缺血/再灌注和缺血预适应过程中对细胞色素c氧化酶的调节作用。我们的工作将集中在由epsilonPKC同工酶调控的PC范式中的一种新的机制事件。更好地了解PC中涉及的分子和细胞内的epsilon PKC靶点将增加对有心肌梗死风险的患者应用心脏保护策略的机会。

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.