Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal
Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal
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DOI：
10.1021/tx049838g
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发表时间：
2004-11-01
影响因子：
4.1
通讯作者：
Petersen, DR
中科院分区：
文献类型：
--
作者：
Carbone, DL;Doorn, JA;Petersen, DR
蛋白质组学方法应用于大鼠肝脏胞质溶胶喂养的饮食组成的高脂肪和乙醇，以确定4-羟基-2-壬烯醛（4-HNE）-修饰的蛋白质在体内。胞质Hsp 72，Hsp 70热休克蛋白家族的诱导型变体，始终是由4-HNE修饰的蛋白质之一。尽管1.3倍的诱导热休克蛋白72在乙醇喂养的动物的肝脏中，没有增加热休克蛋白70介导的荧光素酶重折叠在分离的肝细胞中观察到，这表明抑制这一进程的4-HNE。在补充有重组Hsp 72的兔网织红细胞裂解物（RRL）中观察到荧光素酶重折叠效率分别降低50%和75%，所述重组Hsp 72已在体外分别用10和100 μ M 4-HNE修饰。这一观察结果伴随着25%和50%的伴侣蛋白底物结合相同的治疗后，然而，没有影响之间的复合物形成热休克蛋白72和它的共伴侣蛋白热休克蛋白40观察。用10和100 μ M 4-HNE处理的Hsp 72的胰蛋白酶消化和质谱分析一致地鉴定了在伴侣蛋白的ATP酶结构域中的Cys 267处的加合物形成。通过使用DnaK（一种缺乏Cys 267的细菌Hsp 70变体）证明了该残基在观察到的抑制中的作用。DnaK对4-HNE失活具有抗性。此外，热休克蛋白72是由硫醇不反应的醛丙二醛（MDA）的失活，进一步支持的作用，半胱氨酸在热休克蛋白72抑制4-HNE。最后，用10和100 μ M 4-HNE处理后，Hsp 72对ATP的亲和力分别降低了32%和72%。在慢性酒精性肝损伤模型中，热休克蛋白72的诱导并不伴随着蛋白质重折叠能力的增加。这可能是Hsp 72 ATP酶结构域的4-HNE修饰的结果。
A proteomic approach was applied to liver cytosol from rats fed a diet consisting of high fat and ethanol to identify 4-hydroxy-2-nonenal (4-HNE)-modified proteins in vivo. Cytosolic Hsp72, the inducible variant of the Hsp70 heat shock protein family, was consistently among the proteins modified by 4-HNE. Despite 1.3-fold induction of Hsp72 in the livers of ethanol-fed animals, no increase in Hsp70-mediated luciferase refolding in isolated heptocytes was observed, suggesting inhibition of this process by 4-HNE. A 50% and 75% reduction in luciferase refolding efficiency was observed in rabbit reticulocyte lysate (RRL) supplemented with recombinant Hsp72 which had been modified in vitro with 10 and 100 muM 4-HNE, respectively. This observation was accompanied by a 25% and 50% decrease in substrate binding by the chaperone following the same treatment; however, no effect on complex formation between Hsp72 and its co-chaperone Hsp40 was observed. Trypsin digest and mass spectral analysis of Hsp72 treated with 10 and 100 muM 4-HNE consistently identified adduct formation at Cys267 in the ATPase domain of the chaperone. The role of this residue in the observed inhibition was demonstrated through the use of DnaK, a bacterial Hsp70 variant lacking Cys267. DnaK was resistant to 4-HNE inactivation. Additionally, Hsp72 was resistant to inactivation by the thiol-unreactive aldehyde malondialdehyde (MDA), further supporting a role for Cys in Hsp72 inhibition by 4-HNE. Finally, the affinity of Hsp72 for ATP was decreased 32% and 72% following treatment of the chaperone with 10 and 100 muM 4-HNE, respectively. In a model of chronic alcoholic liver injury, induction of Hsp72 was not accompanied by an increase in protein refolding ability. This is likely the result of 4-HNE modification of the Hsp72 ATPase domain.