Cardioprotective Mechanisms of Glutathione S-transferase P

描述（由申请人提供）：谷胱甘肽S-转移酶（GST）是一个广泛表达的酶超家族，可将还原型谷胱甘肽（GSH）与有毒亲电试剂结合。GST同种型也显示通过蛋白质-蛋白质相互作用调节ASK-1和JNK活化。在人群中，GST亚型以种族和性别特异性方式分布。GST在心血管组织中表达，但GST在心血管生理和病理中的作用尚未研究。我们认为GSTP是心肌中含量最丰富的一种同工型，是心肌防御氧化应激的重要组成部分，在缺血再灌注（I/R）过程中可保护心脏免受急性氧化损伤。为了检验这一假设，我们将评估GSTP对I/R原位期间心脏保护的贡献（目的1）、脂质过氧化衍生的醛（丙烯醛和4-羟基-反式-2-壬烯醛; HNE）的代谢（目的2）和醛解毒（目的3），所述醛是该酶的优选内源性底物。作为功能丧失试验，将GST-敲除小鼠暴露于I/R，而在功能获得试验中，将转基因（TG）小鼠和与心脏限制性表达人多态性GST 1 -1基因的敲除小鼠杂交的TG小鼠暴露于I/R并测定梗死。使用示踪动力学和质谱分析，我们将测量在何种程度上谷胱甘肽占这些醛的代谢之前，期间和之后的心脏缺血（目的2）。为了描述GSTP的贡献，我们将比较GSTP WT、null和TG小鼠心脏中的代谢途径，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缀合、减少醛形成、减少谷胱甘肽-蛋白质加合物、减少过氧化物形成、减少氧化翻译后蛋白质修饰（例如，次磺酸通过谷胱甘肽化，保护心肌GSH水平。此外，我们将确定GSTP是否调节JNK激活和JNK信号传导，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一种新的机制，GSTP的心脏保护的鉴定，这可能是有用的，在评估相对缺血易感性的人群多态性GSTP和开发更有针对性的抗缺血干预措施。 谷胱甘肽S-转移酶（GST）是广泛表达的酶，可使反应性化合物解毒。在人群中，GST以种族和性别特异性方式分布，GST多态性与心血管疾病风险增加相关。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一种新的机制，GSTP的心脏保护的鉴定，这可能是有用的，在了解人群的相对易感性GSTP多态性冠心病和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.