Wood D E, Thomas A, Devi L A, Berman Y, Beavis R C, Reed J C, Newcomb E W
Department of Pathology, New York University Medical Center and Kaplan Comprehensive Cancer Center, New York 10016, USA.
Oncogene. 1998 Sep 3;17(9):1069-78. doi: 10.1038/sj.onc.1202034.
The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.
抗凋亡分子Bcl-2定位于线粒体膜、内质网膜以及核膜。虽然其定位尚未得到严格界定，但促凋亡分子Bax似乎主要是一种胞质蛋白，在凋亡诱导时转位至线粒体。在此，我们在HL-60细胞富含线粒体的膜组分中鉴定出一种蛋白酶活性，该活性能够切割Bax，而胞质组分中不存在这种活性。Bax蛋白酶活性在体外被包括E-64在内的半胱氨酸蛋白酶抑制剂所阻断，这使其有别于所有已知的半胱天冬酶和颗粒酶B，后两者均参与凋亡过程。蛋白酶活性也被针对钙激活中性半胱氨酸内肽酶钙蛋白酶的抑制剂所阻断。通过柱色谱法从HL-60细胞膜组分中部分纯化Bax蛋白酶活性，结果显示一种类似钙蛋白酶的活性是负责切割Bax的蛋白酶。此外，纯化的钙蛋白酶以钙依赖方式切割Bax。用特异性钙蛋白酶抑制剂钙肽素预处理HL-60细胞可有效阻断药物诱导的Bax切割和钙蛋白酶激活，但不影响聚（ADP-核糖）聚合酶切割或细胞死亡。这些结果表明，钙蛋白酶和半胱天冬酶在药物诱导的凋亡过程中被激活，并且钙蛋白酶与半胱天冬酶一起，可能通过选择性作用于细胞底物来参与调节细胞死亡。
