Abrin triggers cell death by inactivating a thiol-specific antioxidant protein

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.