An epsilon PKC interacting protein in preconditioning

描述（由申请人提供）：已明确将ε蛋白激酶C（epsilonPKC）同工酶确定为心脏预处理（PC）的关键介质。因此，研究epsilonPKC选择性信号传导的分子机制以更好地理解其如何介导保护并确定开发PC的epsilonPKC选择性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，在心肌细胞颗粒细胞组分中发现的约18 kDa蛋白的体外磷酸化可用作epsilonPKC激活和epsilonPKC介导的PC的标志物。肽质谱和免疫沉淀分析已经确定，这种蛋白质是细胞色素c氧化酶（COIV）的IV亚基。此外，这种蛋白质可能是新生儿心肌细胞（NCMs）中epsilonPKC的体内底物。我们假设COIV在心肌缺血保护中起着关键的和以前未被描述的作用。以前，我们证明了3 nM 4-β PMA处理优先激活NCM中的epsilonPKC同工酶。在这个建议中，我们将确定是否epsilonPKC可以调节细胞色素c活性，并将这些发现与心脏PC。我们将从接受3 nM 4-β PMA处理和缺氧PC的NMCs中分离线粒体和亚线粒体组分，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。将对从经受缺血性PC的成年大鼠心脏中分离的线粒体和线粒体组分进行类似的分析。本研究的具体目标是：目的i：确定epsilonPKC对新生心肌细胞细胞色素c氧化酶的影响。目的：研究epsilonPKC在新生大鼠心肌细胞预适应中对细胞色素c氧化酶的调节作用。目的：研究epsilonPKC在缺血/再灌注及缺血预适应对成年大鼠心肌细胞色素c氧化酶的调节作用。我们的工作将集中在一个新的机制事件在PC范式调制的epsilonPKC同工酶。 更好地了解参与PC的分子和细胞epsilonPKC靶点将提高心肌梗死风险患者的心脏保护策略的治疗应用的机会。

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.