Morishima Nobuhiro, Nakanishi Keiko, Takenouchi Hiromi, Shibata Takehiko, Yasuhiko Yukuto
Bioarchitect Research Group, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2002 Sep 13;277(37):34287-94. doi: 10.1074/jbc.M204973200. Epub 2002 Jul 3.
Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.
内质网（ER）损伤可特异性诱导无活性的半胱天冬酶 - 12（procaspase - 12）激活为半胱天冬酶 - 12（Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98 - 103），然而半胱天冬酶 - 12激活的功能后果尚不清楚。我们已经表明，重组半胱天冬酶 - 12在胞质提取物中特异性切割并激活无活性的半胱天冬酶 - 9（procaspase - 9）。激活的半胱天冬酶 - 9催化无活性的半胱天冬酶 - 3（procaspase - 3）的切割，这可被半胱天冬酶 - 9特异性抑制剂抑制。尽管人们认为在细胞凋亡过程中，从线粒体释放的细胞色素c是半胱天冬酶 - 9激活所必需的（Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405 - 413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479 - 489），但在内质网应激下，小鼠成肌细胞的无细胞色素c的胞质溶胶中，半胱天冬酶 - 9以及半胱天冬酶 - 12和 - 3均被激活。这些结果表明，半胱天冬酶 - 12可在不涉及细胞色素c的情况下激活半胱天冬酶 - 9。为了研究半胱天冬酶 - 12在下游半胱天冬酶激活中的作用，我们使用了一种在酵母双杂交筛选中鉴定出的半胱天冬酶 - 12结合蛋白来调节半胱天冬酶 - 12的激活。该结合蛋白在体外保护无活性的半胱天冬酶 - 12不被加工。该结合蛋白的稳定表达使无活性的半胱天冬酶 - 12对内质网应激不敏感，从而抑制细胞凋亡以及半胱天冬酶 - 9和 - 3的激活。这些数据表明，无活性的半胱天冬酶 - 9是半胱天冬酶 - 12的底物，并且内质网应激以不依赖细胞色素c的方式触发了一个涉及半胱天冬酶 - 12、 - 9和 - 3的特异性级联反应。
