Activation of Heat Shock Factor by Alkylating Agents Is Triggered by Glutathione Depletion and Oxidation of Protein Thiols (*)
Activation of Heat Shock Factor by Alkylating Agents Is Triggered by Glutathione Depletion and Oxidation of Protein Thiols (*)
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DOI：
10.1074/jbc.271.9.4805
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发表时间：
1996-03
期刊：
影响因子：
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通讯作者：
Hong Liu;R. Lightfoot;J. Stevens
中科院分区：
文献类型：
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作者：
Hong Liu;R. Lightfoot;J. Stevens
热休克蛋白基因的转录激活是蛋白毒性应激的常见反应。许多形成反应性亲电体的药物和化学品通过共价结合亲核官能团来修饰蛋白质结构。虽然这些试剂中的许多也激活hsp 70基因家族的诱导型成员的转录，但尚不清楚细胞蛋白本身的共价修饰是否足够。碘乙酰胺（IDAM）是诱导hsp 70转录的典型烷化剂。然而，IDAM诱导的细胞死亡通过谷胱甘肽的消耗、氧化应激的诱导和脂质过氧化的增加与蛋白质烷基化间接相关。因此，我们确定这些继发性细胞毒性事件中的任何一个是否可能导致hsp 70转录的激活。IDAM处理通过激活热休克转录因子-1（HSF 1）增加hsp 70的转录。抗氧化剂和铁或钙螯合剂的加入阻止了细胞死亡，但没有阻止热休克蛋白70转录或HSF 1激活。然而，蛋白质合成抑制剂放线菌酮阻断低浓度的IDAM激活的热休克蛋白70。此外，在IDAM去除后添加二硫苏糖醇（DTT）阻断hsp 70转录和HSF 1活化而不改变IDAM结合。DTT对高温激活HSF 1无影响。IDAM处理后，细胞的非蛋白质和蛋白质硫醇已下降到低于20%和70%，分别在控制细胞的值。DTT原位处理防止了细胞蛋白硫醇的损失，并阻止了高分子量蛋白质聚集体的形成。因此，蛋白质的烷基化不足以激活hsp 70转录和HSF 1的DNA结合。然而，细胞巯基-二硫键氧化还原状态和二硫键连接的细胞蛋白质聚集体的形成与HSF 1活化和hsp 70转录活化有关。
Transcriptional activation of heat shock protein genes is a common response to proteotoxic stress. Many drugs and chemicals that form reactive electrophiles modify protein structure by binding covalently to nucleophilic functional groups. Although many of these agents also activate transcription of the inducible member of the hsp70 gene family, it is not clear if covalent modification of cellular proteins per se is sufficient. Iodoacetamide (IDAM) is a prototypical alkylating toxicant that induces hsp70 transcription. However, IDAM-induced cell death is indirectly linked to protein alkylation through depletion of glutathione, induction of oxidative stress, and increased lipid peroxidation. Therefore, we determined if any of these secondary cytotoxic events might lead to activation of hsp70 transcription. IDAM treatment increased hsp70 transcription by activating heat shock transcription factor-1 (HSF1). The addition of antioxidants and iron or calcium chelators prevented cell death but did not prevent hsp70 transcription or HSF1 activation. However, the protein synthesis inhibitor cycloheximide blocked activation of hsp70 by low concentrations of IDAM. Furthermore, the addition of dithiothreitol (DTT) after IDAM removal blocked hsp70 transcription and HSF1 activation without altering IDAM binding. DTT had no effect on activation of HSF1 by hyperthermia. After IDAM treatment, cellular nonprotein and protein thiols had decreased to less than 20 and 70%, respectively, of the value in control cells. DTT treatment in situ prevented the loss of cellular protein thiols and blocked the formation of high molecular weight protein aggregates. Thus, alkylation of proteins is insufficient to activate hsp70 transcription and DNA binding of HSF1. However, cellular thiol-disulfide redox status and formation of disulfide linked aggregates of cellular proteins are linked to HSF1 activation and hsp70 transcriptional activation.