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Toxins 2016, 8(11), 321; doi:10.3390/toxins8110321

Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus

1
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland
2
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Lodz 93-232, Poland
3
BioInfoBank Institute, Sw. Marcin 80/82 r.355, Poznan 61-809, Poland
*
Author to whom correspondence should be addressed.
Academic Editors: David J. Fitzgerald and Tomas Girbes
Received: 19 July 2016 / Revised: 1 November 2016 / Accepted: 2 November 2016 / Published: 10 November 2016
(This article belongs to the Collection Immunotoxins 2016)
View Full-Text   |   Download PDF [4651 KB, uploaded 10 November 2016]   |  

Abstract

Exotoxin A (PE) from Pseudomonas aeruginosa is a bacterial ADP-ribosyltransferase, which can permanently inhibit translation in the attacked cells. Consequently, this toxin is frequently used in immunotoxins for targeted cancer therapies. In this study, we propose a novel modification to PE by incorporating the NLS sequence at its C-terminus, to make it a selective agent against fast-proliferating cancer cells, as a nucleus-accumulated toxin should be separated from its natural substrate (eEF2) in slowly dividing cells. Here, we report the cytotoxic activity and selected biochemical properties of newly designed PE mutein using two cellular models: A549 and HepG2. We also present a newly developed protocol for efficient purification of recombinant PE and its muteins with very high purity and activity. We found that furin cleavage is not critical for the activity of PE in the analyzed cell lines. Surprisingly, we observed increased toxicity of the toxin accumulated in the nucleus. This might be explained by unexpected nuclease activity of PE and its potential ability to cleave chromosomal DNA, which seems to be a putative alternative intoxication mechanism. Further experimental investigations should address this newly detected activity to identify catalytic residues and elucidate the molecular mechanism responsible for this action. View Full-Text
Keywords: Exotoxin A; nuclear localization signal; cytotoxicity; nuclease; cancer Exotoxin A; nuclear localization signal; cytotoxicity; nuclease; cancer
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Borowiec, M.; Gorzkiewicz, M.; Grzesik, J.; Walczak-Drzewiecka, A.; Salkowska, A.; Rodakowska, E.; Steczkiewicz, K.; Rychlewski, L.; Dastych, J.; Ginalski, K. Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus. Toxins 2016, 8, 321.

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