Takahashi A, Hirata H, Yonehara S, Imai Y, Lee K K, Moyer R W, Turner P C, Mesner P W, Okazaki T, Sawai H, Kishi S, Yamamoto K, Okuma M, Sasada M
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Japan.
Oncogene. 1997 Jun 12;14(23):2741-52. doi: 10.1038/sj.onc.1201131.
The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.
凋亡的哺乳动物细胞中多种白细胞介素-1β转化酶相关蛋白酶(半胱天冬酶)的激活引发了这样的问题:多种活性半胱天冬酶在凋亡执行过程中是否具有不同作用,以及这些蛋白酶在凋亡信号通路中是如何组织的。在此,我们使用一种亲和标记剂YV(bio)KD-aomk来研究凋亡细胞死亡过程中被激活的半胱天冬酶。YV(bio)KD-aomk在Fas刺激的Jurkat T细胞中鉴定出六种与活性半胱天冬酶相对应的不同多肽。在用星形孢菌素处理时,检测到四种多肽。竞争实验表明,标记的半胱天冬酶具有不同的底物偏好。在每个细胞死亡事件中标记的半胱天冬酶逐步出现,这与激活的半胱天冬酶被组织成蛋白酶级联反应的观点一致。此外,我们发现,通过将非凋亡的Jurkat细胞提取物暴露于半胱天冬酶-8(MACH/FLICE/Mch5)可触发类似于Fas连接诱导的半胱天冬酶的逐步激活。相反,CrmA蛋白,一种Fas诱导凋亡的病毒抑制剂,抑制了半胱天冬酶-8的蛋白酶活性。总体而言,这些发现提供了证据,表明半胱天冬酶-8,一种对CrmA敏感的蛋白酶,负责启动Fas刺激细胞中多种半胱天冬酶的逐步激活。