Cardioprotective Mechanisms of Glutathione S-transferase P

描述(申请人提供)：谷胱甘肽S转移酶(GST)是一个普遍表达的超家族酶，它连接还原型谷胱甘肽和有毒的亲电体。GST亚型也被证明通过蛋白质-蛋白质相互作用来调节ASK-1和JNK的激活。在人类群体中，GST亚型以种族和性别特有的方式分布。GST在心血管组织中有表达，但GST在心血管生理和病理中的作用尚未被研究。我们认为，GSTP是最丰富的心脏亚型，是心肌抵抗氧化应激的重要组成部分，它可以保护心脏免受缺血再灌注(I/R)时的急性氧化损伤。为了验证这一假设，我们将评估GSTP在原位I/R(目标1)、脂质过氧化衍生的醛(丙烯醛和4-羟基-反式-2-壬烯醛；HNE)代谢(该酶的首选内源底物)(目标2)和醛解毒(目标3)过程中对心脏保护的贡献。作为功能丧失测试，GSTP基因缺失的小鼠将暴露于I/R，而在功能测试的获得中，与心脏受限的人GSTP1-1基因表达缺失小鼠杂交的转基因(TG)和转基因小鼠将暴露于I/R并确定脑梗塞。利用示踪动力学和质谱分析，我们将测量在心脏缺血之前、期间和之后这些醛的代谢在多大程度上是由谷胱甘肽引起的(目标2)。为了描述GSTP的贡献，我们将比较GSTP WT、NULL和TG小鼠心脏的代谢途径，包括醛结合和过氧化物酶活性。在目标3中，我们将确定GSTP是否通过增加醛的谷胱甘肽结合，减少醛的形成，减少醛-蛋白质加合物，减少过氧化氢的形成，减少谷胱甘肽对翻译后蛋白的氧化修饰(如磺酸)，以及保护心肌GSH水平来保护心脏免受I/R诱导的损伤。此外，我们将确定GSTP是否调节JNK激活和JNK信号，从而研究GSTP在蛋白质-蛋白质相互作用中的非催化作用。这些研究的成功完成可能导致确定GSTP保护心脏的新机制，这可能有助于评估GSTP基因多态性人群的相对缺血易感性，并有助于开发更有针对性的抗缺血干预措施。与公共健康相关谷胱甘肽S转移酶(GSTs)是一种广泛表达的酶，可解毒活性化合物。在人类群体中，GST以种族和性别特有的方式分布，GST基因多态与心血管疾病风险增加相关。这项研究将评估GSTP是否是防止心脏病发作损伤的心肌防御的重要组成部分。这些研究的成功完成可能导致确定GSTP保护心脏的新机制，这可能有助于了解GSTP基因多态性人群对冠心病的相对易感性，并有助于开发更有针对性的心脏保护干预措施。

DESCRIPTION (provided by applicant): The glutathione S-transferases (GSTs) are a superfamily of ubiquitously expressed enzymes that conjugate reduced glutathione (GSH) with toxic electrophiles. The GST isoforms have also been show to regulate ASK-1 and JNK activation via protein-protein interactions. In human populations, the GST isoforms are distributed in a race- and gender-specific manner. The GSTs are expressed in cardiovascular tissues, but the role of GST in cardiovascular physiology and pathology has not been studied. We propose that GSTP, the most abundant cardiac isoform, is an essential component of myocardial defense against oxidative stress and it protects the heart from acute oxidative damage during ischemia-reperfusion (I/R). To test this hypothesis, we will assess the contribution of GSTP to cardiac protection during I/R in situ (Aim 1), metabolism of lipid peroxidation- derived aldehydes (acrolein and 4-hydroxy-trans-2-nonenal; HNE), which are the preferred endogenous substrates of this enzyme (Aim 2), and aldehyde detoxication (Aim 3). As a loss of function test, GSTP-null mice will be exposed to I/R, while in gain of function tests, transgenic (TG) and TG mice crossed with null mice with cardiac-restricted expression of human polymorphic GSTP1-1 genes will be exposed to I/R and infarction determined. Using tracer kinetics and mass spectrometric analysis, we will measure the extent to which glutathiolation accounts for the metabolism of these aldehydes before, during and after ischemia in the heart (Aim 2). To delineate the contribution of GSTP, we will compare metabolic pathways, including aldehyde conjugation and peroxidase activity, in hearts isolated of GSTP WT, null, and TG mice. In Aim 3, we will determine whether GSTP protects the heart from I/R-induced injury by increasing glutathione conjugation of aldehydes, decreasing aldehyde formation, decreasing aldehyde-protein adducts, decreasing peroxide formation, decreasing oxidative post-translation protein modifications (e.g., sulfenic acid) by glutathiolation, and protecting myocardial GSH level. In addition, we will determine if GSTP regulates JNK activation and JNK signaling thereby investigating a non-catalytic role of GSTP in protein-protein interactions. Successful completion of these studies may lead to the identification of a novel mechanism of cardioprotection by GSTP, which may be useful in assessing the relative ischemic susceptibility of human populations polymorphic in GSTP and in developing more targeted anti-ischemic interventions. PUBLIC HEALTH RELEVANCE The glutathione S-transferases (GSTs) are widely expressed enzymes that detoxify reactive compounds. In human populations, the GSTs are distributed in a race- and gender-specific manner, and GST polymorphisms are associated with increased cardiovascular disease risk. This study will assess if GSTP is an essential component of myocardial defense against injury of heart attacks. Successful completion of these studies may lead to the identification of a novel mechanism of cardioprotection by GSTP, which may be useful in understanding the relative susceptibility of human populations polymorphic in GSTP to coronary heart disease and in developing more targeted cardioprotective interventions.