Dorstyn L, Kumar S
Department of Haematology, Hanson Institute, IMVS, Adelaide, SA, Australia.
Cell Death Differ. 2008 Mar;15(3):461-70. doi: 10.1038/sj.cdd.4402288. Epub 2007 Dec 14.
The activation of caspases is the principal event in the execution of apoptosis. Initiator caspases are activated through an autocatalytic mechanism often involving dimerisation or oligomerisation. In Drosophila, the only initiator caspase DRONC, is tightly inhibited by DIAP1 and removal of DIAP1 permits activation of DRONC by the Drosophila Apaf-1-related killer, ARK. ARK is proposed to facilitate DRONC oligomerisation and autoprocessing at residue E352. This study examines whether autoprocessing of DRONC is required for its activation and for DRONC-mediated cell death. Using purified recombinant proteins, we show here that while DRONC autocleaves at residue E352, mutation of this site did not abolish enzyme activation, DRICE-induced cleavage of DRONC or DRONC-mediated activation of DRICE. We performed a detailed mutational analysis of DRONC cleavage sites and show that overexpression of DRONC cleavage mutants in Drosophila cells retain pro-apoptotic activity. Using an in vitro cell-free assay, we found ARK alone did not activate DRONC and demonstrate a requirement for an additional cytosolic factor in ARK-mediated DRONC activation. These results suggest that, similar to mammalian caspase-2 and caspase-9, the initial cleavage of DRONC is not essential for its activation and suggest a mechanism of ARK-mediated DRONC activation different from that proposed previously.
半胱天冬酶的激活是细胞凋亡执行过程中的主要事件。起始半胱天冬酶通过通常涉及二聚化或寡聚化的自催化机制被激活。在果蝇中，唯一的起始半胱天冬酶DRONC受到DIAP1的严格抑制，去除DIAP1可使果蝇Apaf-1相关杀手ARK激活DRONC。有人提出ARK促进DRONC在E352位点的寡聚化和自加工。本研究探讨了DRONC的自加工对于其激活以及DRONC介导的细胞死亡是否必要。使用纯化的重组蛋白，我们在此表明，虽然DRONC在E352位点自切割，但该位点的突变并未消除酶的激活、DRICE诱导的DRONC切割或DRONC介导的DRICE激活。我们对DRONC切割位点进行了详细的突变分析，并表明在果蝇细胞中过表达DRONC切割突变体保留了促凋亡活性。使用体外无细胞测定法，我们发现单独的ARK不能激活DRONC，并证明在ARK介导的DRONC激活中需要一种额外的胞质因子。这些结果表明，与哺乳动物半胱天冬酶-2和半胱天冬酶-9类似，DRONC的初始切割对其激活并非必不可少，并提出了一种与先前提出的不同的ARK介导的DRONC激活机制。
