Cardioprotective Mechanisms of Glutathione S-transferase P

描述（由申请人提供）：谷胱甘肽s -转移酶（GSTs）是一个普遍表达的酶超家族，它将还原性谷胱甘肽（GSH）与有毒的亲电试剂结合。GST同种异构体也被证明通过蛋白-蛋白相互作用调节ASK-1和JNK的激活。在人类群体中，GST同种异构体以种族和性别特定的方式分布。GST在心血管组织中表达，但GST在心血管生理病理中的作用尚未被研究。我们认为，GSTP是最丰富的心脏异构体，是心肌防御氧化应激的重要组成部分，它保护心脏免受缺血-再灌注（I/R）期间的急性氧化损伤。为了验证这一假设，我们将评估GSTP在原位I/R （Aim 1）、脂质过氧化衍生醛（丙烯醛和4-羟基反式-2-壬烯醛；HNE）的代谢（Aim 2）和醛解毒（Aim 3）期间对心脏保护的贡献。作为功能丧失试验，gstp缺失小鼠将暴露于I/R，而在功能获得试验中，转基因（TG）和TG小鼠与心脏受限表达人类多态GSTP1-1基因的缺失小鼠杂交，将暴露于I/R并确定梗死。使用示踪动力学和质谱分析，我们将测量谷胱甘肽在心脏缺血之前、期间和之后对这些醛代谢的影响程度（目的2）。为了描述GSTP的作用，我们将比较GSTP WT、null和TG小鼠分离心脏的代谢途径，包括醛偶联和过氧化物酶活性。在Aim 3中，我们将确定GSTP是否通过增加谷胱甘肽与醛的结合、减少醛的形成、减少醛-蛋白加合物、减少过氧化物的形成、通过谷胱甘肽化减少氧化翻译后蛋白修饰（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会导致GSTP保护心脏的新机制的确定，这可能有助于评估GSTP多态性人群的相对缺血易感性，并开发更有针对性的抗缺血干预措施。谷胱甘肽s -转移酶（GSTs）是一种广泛表达的酶，可对活性化合物进行解毒。在人群中，GST以特定种族和性别的方式分布，GST多态性与心血管疾病风险增加有关。本研究将评估GSTP是否是心肌防御心脏病发作损伤的重要组成部分。这些研究的成功完成可能会导致GSTP对心脏保护的新机制的鉴定，这可能有助于了解GSTP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.