Jürgensmeier J M, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed J C
Program on Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4997-5002. doi: 10.1073/pnas.95.9.4997.
Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.
Bax是Bcl-2蛋白家族的促凋亡成员，定位于线粒体外膜。Bax是直接通过其作为通道蛋白的假定功能促进细胞死亡，还是通过抑制细胞死亡蛋白酶（半胱天冬酶）的细胞调节因子间接促进细胞死亡，这存在争议。我们在此表明，向分离的线粒体中添加亚微摩尔量的重组Bax蛋白可诱导细胞色素c（Cyt c）释放，而代表Bax BH3结构域的肽则无活性。当置于纯化的胞质溶胶中时，线粒体和Bax单独都不会诱导半胱天冬酶的蛋白水解加工和激活。相反，Bax和线粒体的组合触发了Cyt c从线粒体的释放，并在胞质溶胶中诱导了半胱天冬酶的激活。经Bax处理的线粒体的上清液也诱导了半胱天冬酶的加工和激活。重组Bcl-XL蛋白消除了Bax诱导的Cyt c从分离的线粒体中的释放，并阻止了半胱天冬酶的激活。相反，广谱特异性半胱天冬酶抑制剂苄氧羰基-缬氨酰-丙氨酰-天冬氨酰-（0-甲基）-氟甲基酮（zVAD-fmk）和半胱天冬酶抑制蛋白X-IAP对体外Bax诱导的Cyt c从线粒体中的释放没有影响，但阻止了胞质提取物中半胱天冬酶的后续激活。与线粒体通透性转换的经典诱导剂Ca2+不同，Bax在体外不会诱导线粒体肿胀。由于通透性转换引起的细胞器肿胀会导致外膜破裂，因此，这些发现将这两个事件分开，这意味着Bax使用另一种机制触发Cyt c从线粒体中释放。
