Liu H, Lightfoot R, Stevens J L
W. Alton Jones Cell Science Center, Lake Placid, New York 12946, USA.
J Biol Chem. 1996 Mar 1;271(9):4805-12.
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.
热休克蛋白基因的转录激活是对蛋白毒性应激的常见反应。许多形成反应性亲电试剂的药物和化学物质通过与亲核官能团共价结合来修饰蛋白质结构。尽管这些试剂中的许多也能激活hsp70基因家族诱导型成员的转录，但尚不清楚细胞蛋白质的共价修饰本身是否足够。碘乙酰胺（IDAM）是一种典型的烷基化毒物，可诱导hsp70转录。然而，IDAM诱导的细胞死亡通过谷胱甘肽耗竭、氧化应激诱导和脂质过氧化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hsp70转录的激活。IDAM处理通过激活热休克转录因子-1（HSF1）增加hsp70转录。添加抗氧化剂以及铁或钙螯合剂可防止细胞死亡，但不能阻止hsp70转录或HSF1激活。然而，蛋白质合成抑制剂环己酰亚胺可阻断低浓度IDAM对hsp70的激活。此外，在去除IDAM后添加二硫苏糖醇（DTT）可阻断hsp70转录和HSF1激活，而不会改变IDAM的结合。DTT对热疗激活HSF1没有影响。IDAM处理后，细胞非蛋白质和蛋白质硫醇分别降至对照细胞中该值的20%和70%以下。原位DTT处理可防止细胞蛋白质硫醇的损失，并阻断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hsp70转录和HSF1的DNA结合。然而，细胞硫醇-二硫键氧化还原状态以及细胞蛋白质二硫键连接聚集体的形成与HSF1激活和hsp70转录激活有关。
