Mitochondrial PKC epsilon in cardiac protection

描述（由申请人提供）：一氧化氮（NO）供体通过涉及蛋白激酶C e（PKCe）的信号机制来保护心肌缺血/再灌注损伤。PKCe靶向进行保护的下游效应物仍然不清楚。最近的一项研究表明，腺嘌呤核苷酸转运蛋白（ANT 1）--线粒体通透性转换（MPT）孔的核心单位作为PKC的信号伴侣（。目前研究的重点是确定NO供体，临床相关的心脏保护药物，是否通过MPT孔的ANT 1组分的调节起作用，并检查PKCe在此过程中直接修饰孔的作用。该研究将采用标准的线粒体功能测定，以测量MPT孔活性，标准的生物化学程序，以确定蛋白质表达和相互作用，以及最先进的质谱法，以检查PKC对ANT 1的磷酸化。中心假设是，在NO供体诱导的心脏保护中，PKCe的关键任务是降低线粒体通透性转换的倾向。该作用部分涉及MPT孔的核心组分ANT 1的直接修饰。具体目标是：（1）探讨PKCe依赖性调节线粒体通透性转换在NO诱导的小鼠心肌保护模型中的作用。(2)确定MPT孔的必需元件ANT 1是否是PKC的下游分子靶点（并鉴定在NO诱导的心脏保护过程中被PKC Ce磷酸化的ANT 1的特定氨基酸残基）。

DESCRIPTION (provided by applicant): Nitric oxide (NO) donors protect against myocardial ischemia/reperfusion injury via signaling mechanisms that involve protein kinase C e (PKCe). The downstream effectors targeted by PKCe to carry out protection remain poorly defined. A recent study has implicated the adenine nucleotide translocator (ANT1)---a core unit of the mitochondrial permeability transition (MPT) pore as a signaling partner of PKC(. The focus of the current investigation is to determine whether NO donors, clinically relevant cardiac protective drugs, act via modulation of the ANT1 component of the MPT pore and to examine the role of direct modification of the pore by PKCe in this process. The study will employ standard mitochondrial function assays, to measure MPT pore activity, standard biochemistry procedures, to determine protein expression and interactions, and state-of-the-art mass spectrometry, to examine phosphorylation of ANT1 by PKC(. The central hypothesis is that a critical task of PKCe in NO donor-induced cardiac protection is to reduce the propensity for mitochondrial permeability transition. This action involves, in part, direct modification of a core component of the MPT pore, ANT1. The specific aims are: (1) To elucidate the role of PKCe-dependent regulation of mitochondrial permeability transition in a murine model of NO-induced cardiac protection. (2) To determine whether ANT1, an essential element of the MPT pore, is a downstream molecular target of PKC( and to identify the specific amino acid residues of ANT1 phosphorylated by PKCe during NO-induced cardiac protection.