Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation
Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation
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DOI：
10.1074/jbc.m109.095083
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发表时间：
2010-05-28
影响因子：
4.8
通讯作者：
Green, Douglas R.
中科院分区：
文献类型：
--
作者：
Oberst, Andrew;Pop, Cristina;Green, Douglas R.
Caspase-8是一种半胱氨酸蛋白酶，由细胞膜胞浆表面的膜结合受体激活，启动外在的细胞凋亡途径。Caspase-8的激活依赖于非活性单体酶原在激活的受体复合体中的募集，在那里它们产生一个由两个催化域组成的完全活性的酶。虽然使用药物介导的亲和系统或渗透压盐来驱动二聚的体外研究表明，未切割的caspase-8可以很容易地被单独的二聚激活，但使用小鼠模型的体内结果得出了相反的结论。此外，除了结构域间的自动处理外，caspase-8还可以被激活的执行者caspase切割，关于这种切割事件是否会导致caspase-8激活的报道一直相互矛盾。在这里，我们通过在体外和在缺乏内源性caspase-8的细胞系中对在结构域间裂解位点带有禁止突变的caspase-8突变体的激活特性进行研究来解决这些问题，我们发现这些裂解位点的消除排除了前结构域驱动的二聚激活caspase-8的可能性。然后，我们进一步探索caspase-8的域间切割的后果，通过改造烟草蚀刻病毒蛋白酶来创建一个系统，在该系统中，caspase-8的切割和二聚化都可以在活细胞中独立控制。我们发现，与单靠结构域间切割很容易激活的执行者caspase不同，caspase-8的二聚化或切割都不足以激活caspase-8或诱导细胞凋亡，只有酶原的协调二聚化和切割才能在体外产生有效的激活和细胞系统的凋亡。
Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.