| Summary: | 碩士 === 國立臺灣大學 === 生化學研究所 === 89 === Abrin, a toxic protein isolated from the plant Abrus precatorius, is one of the type II RIPs (ribosome-inactivating proteins) and composed of A chain and B chain. The B chain binds to a cell surface receptor, facilitating endocytosis of the whole molecule, while the A chain exhibits an RNA N-glycosidase activity, removing a specific adenine residue (A4324) from the 28S rRNA. This N-glycosidase activity results in loss of protein elongation and presumably the subsequent death of the treated cell.
In this laboratory, it had recently been demonstrated that abrin induces apoptosis by increasing the intracellular reactive oxygen species (ROS) and by releasing cytochrome c from mitochondria to cytosol. To further investigate these intracellular effects, yeast two-hybrid system was used to search for abrin- interacting proteins that may be potentially involved in the cell death pathway induced by abrin. The results showed that a protein designated as antioxidant protein-1 (AOP-1) specifically interacts with abrin A-chain. It was shown that abrin A-chain interacts with AOP-1 and inactivates its antioxidant activity, leading to the appearance of intracellular ROS and hence the occurring of cell death.
To study how ABRA interacts with AOP-1 bringing about apoptosis, we examined the AOP-1 interacting sites of ABRA. There are three domains in ABRA, namely domain I, II, and III (ABRA-I, ABRA-II, and ABRA-III). The various truncated domains were cloned and expressed to obtain the truncated fragments of ABRA. Two different strategies were employed to study the interaction between various ABRA truncated proteins and AOP-1: (1) yeast two-hybrid system, and (2) pull down assay.
The results of yeast two-hybrid assay showed that ABRA-III interacted with AOP-1 resulting in the expression of lacZ and HIS3 reporter genes. The lacZ expression of this combination exhibited a b-galactosidase activity of 160 units, and was fewer than that of ABRA only in 20 units. The lacZ expression of other combination, however, exhibited low activity of b-galactosidase. In in vitro binding assay, ABRA-III also bound to AOP-1 in a similar way like ABRA did, and the AOP-1 binding intensity of ABRA-III was 87.5% of that of ABRA. To investigate the effect of ABRA-III on biological activity of AOP-1, in vitro inhibition assay was carried out to study whether the binding of ABRA-III to AOP-1 could affect the antioxidant activity of AOP-1. It was interesting to find that the presence of ABRA-III only caused 1.1% of AOP-1 inactivation, but the AOP-1 activity decreased dramatically from 80.1% to 50.1% when ABRA-I was present in the reaction mixture. From these results, we proposed that ABRA triggers cell death through binding to AOP-1 by ABRA-III and inhibiting AOP-1 by ABRA-I.
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