Abstract
Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells. Previous studies from our laboratory suggested a link between these two events. Using an active site mutant of abrin A-chain which exhibits 225-fold lower protein synthesis inhibitory activity than the wild-type abrin A-chain, we demonstrate in this study that inhibition of protein synthesis induced by abrin is the major factor triggering unfolded protein response leading to apoptosis. Since abrin A-chain requires the B-chain for internalization into cells, the wild-type and mutant recombinant abrin A-chains were conjugated to native ricin B-chain to generate hybrid toxins, and the toxic effects of the two conjugates were compared. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild-type ricin B-rABRA conjugate as expected. The mutant conjugate activated p38MAPK and caspase-3 similar to its wild-type counterpart although at later time points. Overall, these results confirm that inhibition of protein synthesis is the major event contributing to abrin-mediated apoptosis.
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Acknowledgments
We thank Dr. Omana Joy, Puja Pai, Vasista Adiga and Kavya Ananthaswamy of the FACS facility, IISc for the help provided. The facility is supported by Department of Biotechnology (DBT) and Council for Scientific and Industrial Research (CSIR), Government of India. There are no conflicts of interest to report.
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11010_2015_2355_MOESM1_ESM.tif
Supplementary material 1 (TIFF 178 kb). Comparison of secondary structure of conjugates using circular dichroism. The CD spectra of the proteins (50 µg/ml) in PBS were recorded in the far-UV region as mentioned in materials and methods
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Supplementary material 2 (TIFF 885Â kb). Endocytosis of the conjugates in Jurkat cells. Jurkat cells were treated with 10Â ng/ml concentration of wild type and mutant conjugate for 1Â h. After the treatment cells were fixed, incubated with primary antibody D6F10 followed by incubation with Alexa fluor 633-labelled secondary antibody
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Mishra, R., Kumar, M.S. & Karande, A.A. Inhibition of protein synthesis leading to unfolded protein response is the major event in abrin-mediated apoptosis. Mol Cell Biochem 403, 255–265 (2015). https://doi.org/10.1007/s11010-015-2355-9
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DOI: https://doi.org/10.1007/s11010-015-2355-9