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

本研究计划旨在阐明氧化应激在分离细胞中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）的最佳标志物。HNE是由最丰富的一类n-6多不饱和脂肪酸过氧化生成的醛类终产物。与自由基类似，HNE是一种亲电试剂，易于与蛋白质反应形成HNE-蛋白质加合物，但其相对较长的半衰期使其成为对邻近细胞的损伤传播的候选者。人们对HNE的兴趣不仅源于其作为氧化应激诱导的LPO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。在目前的研究中，我们感兴趣的是验证正常人软骨细胞中hne -蛋白加合物的积累在生理氧化应激下会增加，从而调节细胞活性的假设。首先，我们将使用蛋白质组学技术来识别生理氧化应激下正常人软骨细胞中HNE结合的特异性蛋白质。然后，我们将重点关注与hne结合蛋白积累有关的酶机制。谷胱甘肽- s -转移酶（GST）可以解毒HNE，是细胞中抗氧化应激的主要防御手段。我们将优化分离软骨细胞的化学和分子干预，以便通过GST调节控制hne结合蛋白水平。最后，我们建议澄清hne结合蛋白水平增加对人类正常软骨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.