Protein modification by 4-hydroxynonenal (HNE) modulates cell function: identification of specific proteins as cellular targets of HNE binding

本研究旨在阐明氧化应激在分离细胞蛋白质修饰中的作用。近年来，羟基壬烯醛(HNE)已成为体内氧化应激诱导的脂质过氧化(LPO)最有效的标志物。HNE是最丰富的n-6多不饱和脂肪酸过氧化生成的一种醛的最终产物。与自由基类似，HNE是一种亲电性物质，很容易与蛋白质反应形成HNE-蛋白质加合物，但其相对较长的半衰期使其有可能将损伤传播到邻近细胞。对HNE的兴趣不仅源于它作为氧化应激诱导LPO的生物标志物的潜在用途，还因为越来越多的证据表明HNE能够调节信号通路和蛋白质修饰。在本研究中，我们有兴趣验证这样一种假设，即在生理氧化应激下，正常人类软骨细胞中HNE蛋白加合物的积累会增加，从而调节细胞的活动。首先，我们将使用蛋白质组学技术来确定在生理氧化应激下正常人类软骨细胞中HNE结合的特定蛋白。然后，我们将重点介绍HNE结合蛋白积累所涉及的酶机制。谷胱甘肽-S-转移酶(GST)为HNE解毒，是细胞抵御氧化应激的主要防御机制。我们将优化对分离的软骨细胞的化学和分子干预，以便通过GST调节来控制HNE结合蛋白的水平。最后，我们建议阐明人类正常软骨细胞中HNE结合蛋白水平升高对细胞表型和代谢的意义。

This research program is aimed at clarifying the role of oxidative stress in proteins modification in isolated cells. Recently, hydroxynonenal (HNE) have emerged as the best available markers of oxidative stress-induced lipid peroxidation (LPO) in vivo. HNE is an aldehyde end-product generated by peroxidation of the most abundant class of n-6 polyunsaturated fatty acids. Similar to free radicals, HNE is electrophiles that react readily to proteins to form HNE-protein adducts, but its relatively longer half-life makes it candidates for the propagation of the damage to neighboring cells. The interest for HNE stems not only from its potential use as a biomarker of oxidative stress-induced LPO, but also because of accumulating evidence indicating that HNE is able to modulate signaling pathways and protein modification. In the present study, we are interested to test the hypothesis that the accumulation of HNE-protein adducts in normal human chondrocytes will increase under physiological oxidative stress and consequently modulate cell activities. Firstly, we will use proteomics techniques in order to identify specific proteins which will be targeted by HNE binding in normal human chondrocytes under physiological oxidative stress. We will then focus on the enzymatic mechanism involved in HNE-bound proteins accumulation. The glutathione-S-transferase (GST), which detoxifies HNE, acts as a major defense against oxidative stress in cells. We will optimize the chemical and molecular interventions in isolated chondrocytes in order to control HNE-bound proteins levels through GST regulation. Finally, we propose to clarify the significance of increased levels of HNE-bound proteins on cell phenotype and metabolism in human normal chondrocytes.