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Ce （PKCe）的信号机制保护心肌免受缺血/再灌注损伤。PKCe靶向的下游效应物进行保护仍然不明确。最近的一项研究表明，腺嘌呤核苷酸转运子（ANT1）是线粒体通透性转化（MPT）孔的核心单元，是PKC的信号伙伴。目前研究的重点是确定NO供体，临床相关的心脏保护药物，是否通过调节MPT孔的ANT1成分起作用，并检查PKCe直接修饰孔在这一过程中的作用。该研究将采用标准的线粒体功能分析来测量MPT孔活性，标准的生物化学程序来确定蛋白质表达和相互作用，以及最先进的质谱法来检测PKC（PKC）对ANT1的磷酸化。核心假设是PKCe在NO供体诱导的心脏保护中的一个关键任务是减少线粒体通透性转变的倾向。这一作用部分涉及到MPT孔的核心成分ANT1的直接修饰。具体目的是：(1)阐明pkce依赖性调节线粒体通透性转变在一氧化氮诱导的小鼠心脏保护模型中的作用。(2)确定MPT孔必需元件ANT1是否为PKC的下游分子靶点，并鉴定no诱导心脏保护过程中PKCe磷酸化ANT1的特异性氨基酸残基。

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.