Shih S F, Wu Y H, Hung C H, Yang H Y, Lin J Y
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 10081, Taiwan, Republic of China.
J Biol Chem. 2001 Jun 15;276(24):21870-7. doi: 10.1074/jbc.M100571200. Epub 2001 Apr 2.
Abrin A-chain (ABRA) inhibits protein synthesis by its N-glycosidase activity as well as induces apoptosis, but the molecular mechanism of ABRA-induced cell death has been obscure. Using an ABRA mutant that lacks N-glycosidase activity as bait in a yeast two-hybrid system, a 30-kDa antioxidant protein-1 (AOP-1) was found to be an ABRA(E164Q)-interacting protein. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay. The colocalization of endogenous AOP-1 and exogenous ABR proteins in the cell was demonstrated by confocal immunofluorescence. We also demonstrated that ABRA attenuates AOP-1 antioxidant activity in a dose-dependent manner and the intracellular level of reactive oxygen species (ROS) increases in ABR-treated cells. Moreover, ROS scavengers N-acetylcysteine and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl delayed programmed cell death. This indicates that ROS are important mediators of ABR-induced apoptosis. When ectopically expressed, AOP-1 blocked the release of cytochrome c and prevented apoptosis in ABR-treated cells. These findings suggest that the binding of ABRA to AOP-1 promotes apoptosis by inhibiting the mitochondrial antioxidant protein AOP-1, resulting in the increase of intracellular ROS and the release of cytochrome c from the mitochondria to the cytosol, which activates caspase-9 and caspase-3.
相思子毒素A链（ABRA）通过其N-糖苷酶活性抑制蛋白质合成并诱导细胞凋亡，但ABRA诱导细胞死亡的分子机制一直不清楚。在酵母双杂交系统中，使用缺乏N-糖苷酶活性的ABRA突变体作为诱饵，发现一种30 kDa的抗氧化蛋白-1（AOP-1）是与ABRA（E164Q）相互作用的蛋白。通过谷胱甘肽S-转移酶下拉试验在体外进一步证实了这种相互作用。共聚焦免疫荧光证明了内源性AOP-1和外源性ABR蛋白在细胞中的共定位。我们还证明，ABRA以剂量依赖性方式减弱AOP-1的抗氧化活性，并且在ABR处理的细胞中活性氧（ROS）的细胞内水平增加。此外，ROS清除剂N-乙酰半胱氨酸和4-羟基-2,2,6,6-四甲基哌啶-1-氧基延迟了程序性细胞死亡。这表明ROS是ABR诱导的细胞凋亡的重要介质。当异位表达时，AOP-1阻断细胞色素c的释放并防止ABR处理的细胞发生凋亡。这些发现表明，ABRA与AOP-1的结合通过抑制线粒体抗氧化蛋白AOP-1促进细胞凋亡，导致细胞内ROS增加以及细胞色素c从线粒体释放到细胞质中，从而激活caspase-9和caspase-3。
