Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease
Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease
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DOI：
10.1124/jpet.105.088088
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发表时间：
2005-10-01
影响因子：
3.5
通讯作者：
Petersen, DR
中科院分区：
文献类型：
--
作者：
Carbone, DL;Doorn, JA;Petersen, DR
氧化应激期间的脂质过氧化导致硫醇反应性α、β-不饱和醛的浓度增加，包括4-羟基-2-壬烯醛(4-HNE)和4-氧代-2-壬烯醛(4-ONE)。据记载，这些醛在与特定残基形成加合物后具有破坏蛋白质功能的能力。因此，为了在乙醇诱导的氧化应激模型中鉴定 4-HNE 修饰的蛋白质，将蛋白质组学方法应用于从喂食高脂肪/乙醇组合饮食的大鼠制备的肝脏部分。结果显示，在酒精处理的动物中，必需的 90 kDa 热休克蛋白 (Hsp90) 始终被 4-HNE 修饰。然后使用萤火虫荧光素酶作为客户蛋白进行体外陪伴实验，以评估 4-HNE 修饰对纯化的重组人 Hsp90 的功能影响，该醛的浓度范围为 23 至 450 μM。用 4-ONE 修饰 Hsp90 也导致伴侣蛋白的显着抑制。由于 4-HNE 和 4-ONE 选择性地与 Cys 反应，因此这些数据表明了硫醇特异性的抑制机制。因此，用特定的硫醇修饰剂 N-乙基马来酰亚胺处理 Hsp90 后，证实了硫醇敏感性，导致伴侣分子在浓度低至 6 μM（1:1 M 比例）时失活 99% 以上。最后，对 4-HNE 修饰的 Hsp90 进行胰蛋白酶消化，然后进行液相色谱/串联质谱肽分析，鉴定出 Cys 572 为 4-HNE 修饰的位点。因此，本文提出的结果证实，4-HNE 在酒精诱导的氧化应激大鼠模型中一致修饰 Hsp90，并且该蛋白的陪伴活性会因硫醇修饰而发生失调。
Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive alpha, beta-unsaturated aldehyde, including 4-hydroxy-2-nonenal (4- HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepared from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alcohol-treated animals. In vitro chaperoning experiments using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concentrations of this aldehyde ranging from 23 to 450 mu M. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concentrations as low as 6 mu M (1:1 M ratio). Finally, tryptic digest of 4-HNE- modified Hsp90 followed by liquid chromatography/tandem mass spectrometry peptide analysis identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alcohol-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.