Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation
Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation
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DOI：
10.1074/jbc.m906383199
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Seo, JS
中科院分区：
文献类型：
--
作者：
Li, CY;Lee, JS;Seo, JS
热休克蛋白70(HSP70)是一种细胞凋亡的抑制因子。我们还观察到HSP70在预热的U937和稳定表达HSP70的U937(U937/HSP70)细胞中对凋亡细胞死亡的抑制作用。然而，HSP70阻止细胞凋亡的分子机制仍有待解决。为了解决这个问题，我们在体外系统中研究了HSP70对细胞凋亡过程的影响。加入dATP后，正常细胞胞浆caspase-3裂解和DNA片段化，而预热的U937或U937/HSP70细胞则未检测到。此外，将纯化的重组HSP70加入到正常的胞浆组分中，可以防止caspase-3的裂解和DNA片段化，这表明HSP70阻止了caspase-3加工的上游的细胞凋亡。由于在预热的U937和U937/HSP70细胞中，尽管caspase-3的激活和细胞死亡被阻止，但致死性热休克仍然将细胞色素c从线粒体释放到胞浆中，因此，很明显，HSP70作用于细胞色素c释放的下游。用纯化的HSP70缺失突变体在体外获得的结果表明，羧基1/3区域(从氨基酸438到641)包括多肽结合域和羧基末端的EEVD序列对于阻止caspase-3的加工是必不可少的。根据这些结果，我们认为HSP70在细胞色素c释放的下游和caspase-3的激活的上游发挥着强大的细胞凋亡抑制作用。
Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-over-expressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by Lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.