Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus.
Exotoxin A (PE) fromPseudomonas aeruginosais 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 itsC-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.
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland. m.borowiec@cent.uw.edu.pl.
Marta
M
Borowiec
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Lodz 93-232, Poland. mgorzkiewicz@cbm.pan.pl.
Michal
M
Gorzkiewicz
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland. j.grzesik@cent.uw.edu.pl.
Joanna
J
Grzesik
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Lodz 93-232, Poland. adrzewiecka@cbm.pan.pl.
Aurelia
A
Walczak-Drzewiecka
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Lodz 93-232, Poland. asalkowska@cbm.pan.pl.
Anna
A
Salkowska
BioInfoBank Institute, Sw. Marcin 80/82 r.355, Poznan 61-809, Poland. ewelinarodakowska@bioinfo.pl.
Ewelina
E
Rodakowska
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland. k.steczkiewicz@cent.uw.edu.pl.
Kamil
K
Steczkiewicz
BioInfoBank Institute, Sw. Marcin 80/82 r.355, Poznan 61-809, Poland. leszek@bioinfo.pl.
Leszek
L
Rychlewski
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Lodz 93-232, Poland. jdastych@cbm.pan.pl.
Jaroslaw
J
Dastych
Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland. kginal@cent.uw.edu.pl.
Krzysztof
K
Ginalski
D000072283:A549 Cells; D036002:ADP Ribose Transferases / Q000235:genetics* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D005098:Exotoxins / Q000235:genetics* / Q000633:toxicity*; D056945:Hep G2 Cells; D006801:Humans; D000922:Immunotoxins; D015202:Protein Engineering; D037521:Virulence Factors / Q000235:genetics* / Q000633:toxicity*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
Exotoxin A; cancer; cytotoxicity; nuclear localization signal; nuclease