Zhou Q, Snipas S, Orth K, Muzio M, Dixit V M, Salvesen G S
Burnham Institute, San Diego, California 92037, USA.
J Biol Chem. 1997 Mar 21;272(12):7797-800. doi: 10.1074/jbc.272.12.7797.
When ectopically expressed in animal cells, cytokine response modifier A (CrmA), a product of the cowpox virus, prevents programmed cell death initiated by a variety of stimuli. Since CrmA is a proteinase inhibitor, its target is probably a protease that promotes cell death. The identification of this target is crucial in delineating essential regulation points that modulate the apoptotic program. We have compared the kinetics of interaction of CrmA with five proteases that may play a role in apoptosis. Four of the proteases, all members of the caspase family, are inhibited with widely different rates and affinities ranging over 5 orders of magnitude. One is not inhibited at all under the experimental conditions. CrmA is quite selective in its ability to inhibit caspases, showing the highest affinity for interleukin-1beta-converting enzyme and the second highest for the caspase FLICE (Ki = 0.95 nM), identified as a component of the intracellular signaling complex recruited by ligation of the death receptor Fas. On the basis of comparative inhibitor kinetics, we propose that CrmA is unlikely to inhibit the caspases Yama, Mch2, or LAP3 in vivo but that its inhibition of FLICE is of a magnitude for this protease to be a key target of CrmA during Fas-mediated apoptosis. Therefore, our results support the hypothesis that FLICE catalyzes a crucial step in the promotion of cell death.
当在动物细胞中异位表达时，牛痘病毒的产物细胞因子反应调节因子A（CrmA）可阻止由多种刺激引发的程序性细胞死亡。由于CrmA是一种蛋白酶抑制剂，其靶标可能是一种促进细胞死亡的蛋白酶。鉴定该靶标对于描绘调节凋亡程序的关键调控点至关重要。我们比较了CrmA与五种可能在凋亡中起作用的蛋白酶相互作用的动力学。其中四种蛋白酶，均为半胱天冬酶家族成员，被抑制的速率和亲和力差异很大，范围超过5个数量级。另一种在实验条件下根本不被抑制。CrmA在抑制半胱天冬酶的能力方面具有相当的选择性，对白细胞介素-1β转化酶的亲和力最高，对被鉴定为死亡受体Fas连接所募集的细胞内信号复合物成分的半胱天冬酶FLICE的亲和力次之（Ki = 0.95 nM）。基于比较抑制剂动力学，我们提出CrmA在体内不太可能抑制半胱天冬酶Yama、Mch2或LAP3，但它对FLICE的抑制程度足以使该蛋白酶成为Fas介导的凋亡过程中CrmA的关键靶标。因此，我们的结果支持FLICE催化促进细胞死亡关键步骤的假说。
