Hu Y, Benedict M A, Wu D, Inohara N, Núñez G
Departments of Pathology and Comprehensive Cancer Center, 1500 East Medical Center Drive, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4386-91. doi: 10.1073/pnas.95.8.4386.
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
近期研究表明，秀丽隐杆线虫的CED-4与死亡蛋白酶CED-3相互作用并促进其激活，且这种激活受到CED-9的抑制。在此我们发现，CED-4的哺乳动物同源物Apaf-1能够在哺乳动物细胞中与多种死亡蛋白酶结合，包括caspase-4、caspase-8、caspase-9以及线虫的CED-3。与caspase-9的相互作用由Apaf-1的N端CED-4样结构域介导。Apaf-1的表达增强了caspase-9的杀伤活性，这一过程需要Apaf-1的CED-4样结构域。此外，Apaf-1在体内促进了caspase-9的加工和激活。Bcl-XL是Bcl-2家族的抗凋亡成员，在哺乳动物细胞中它与Apaf-1和caspase-9存在物理相互作用。Apaf-1与Bcl-XL的结合通过其CED-4样结构域以及包含WD-40重复序列的C端结构域介导。Bcl-XL的表达抑制了哺乳动物细胞中Apaf-1与caspase-9的结合。值得注意的是，从大肠杆菌或昆虫细胞中纯化得到的重组Bcl-XL抑制了Apaf-1依赖的caspase-9加工。此外，Bcl-XL未能抑制由组成型活性Apaf-1突变体介导的caspase-9加工，这表明Bcl-XL通过Apaf-1调节caspase-9。这些实验证明Bcl-XL与caspase-9和Apaf-1结合，并表明Bcl-XL抑制了由Apaf-1介导的caspase-9成熟，这一过程从线虫到人类在进化上是保守的。
