Mizuta Takeshi, Shimizu Shigeomi, Matsuoka Yousuke, Nakagawa Takashi, Tsujimoto Yoshihide
Laboratory of Molecular Genetics, Department of Medical Genetics, Osaka University Medical School, Osaka 565-0871, Japan.
J Biol Chem. 2007 Jun 1;282(22):16623-30. doi: 10.1074/jbc.M611060200. Epub 2007 Apr 4.
Bax and Bak are multidomain pro-apoptotic members of the Bcl-2 family of proteins that regulate mitochondria-mediated apoptosis by direct modulation of mitochondrial membrane permeability. Since double-knock-out mouse embryonic fibroblasts with deficiency of Bax and Bak are resistant to multiple apoptotic stimuli, Bax and Bak are considered to be an essential gateway for various apoptotic signals. Here we showed that the combination of calcium ionophore A23187 and arachidonic acid induced cytochrome c release and caspase-dependent death of double-knock-out mouse embryonic fibroblasts, indicating that other mechanisms of cytochrome c release exist. Furthermore, A23187/arachidonic acid (ArA)-induced caspase-dependent death was significantly suppressed by the treatment of several serine protease inhibitors including 4-(2-aminoethyl)benzenesulfonylfluoride and l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone but not the overexpression of anti-apoptotic Bcl-2 family of proteins or the inhibition of mitochondrial membrane permeability transition. These results indicate that there are at least two mechanisms of cytochrome c release leading to caspase activation, a Bax/Bak-dependent mechanism and a Bax/Bak-independent, but serine protease(s)-dependent, mechanism.
Bax和Bak是Bcl-2蛋白家族的多结构域促凋亡成员，它们通过直接调节线粒体膜通透性来调控线粒体介导的细胞凋亡。由于缺乏Bax和Bak的双敲除小鼠胚胎成纤维细胞对多种凋亡刺激具有抗性，因此Bax和Bak被认为是各种凋亡信号的重要通道。在此我们表明，钙离子载体A23187和花生四烯酸的组合可诱导双敲除小鼠胚胎成纤维细胞释放细胞色素c并引发caspase依赖性死亡，这表明存在细胞色素c释放的其他机制。此外，包括4-(2-氨基乙基)苯磺酰氟和l-1-氯-3-(4-甲苯磺酰胺基)-4-苯基-2-丁酮在内的几种丝氨酸蛋白酶抑制剂处理可显著抑制A23187/花生四烯酸（ArA）诱导的caspase依赖性死亡，但抗凋亡Bcl-2蛋白家族的过表达或线粒体膜通透性转换的抑制则不能。这些结果表明，至少存在两种导致caspase激活的细胞色素c释放机制，一种是Bax/Bak依赖性机制，另一种是Bax/Bak非依赖性但丝氨酸蛋白酶依赖性机制。
