Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus
Marta Borowiec1, Michal Gorzkiewicz2, Joanna Grzesik3
1Laboratory 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.
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.
Insights
Pseudomonas aeruginosa exotoxin A (PE) was modified with a nuclear localization sequence (NLS) for targeted cancer therapy. This engineered toxin showed increased cytotoxicity, potentially via a novel DNA-cleaving mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Exotoxin A (PE) from Pseudomonas aeruginosa is an ADP-ribosyltransferase that inhibits protein translation.
- PE is utilized in immunotoxins for targeted cancer therapy.
- Targeting cancer cells requires selective agents that differentiate from healthy cells.
Purpose of the Study:
- To engineer a novel PE mutein by incorporating a nuclear localization sequence (NLS) at its C-terminus.
- To evaluate the cytotoxic activity and biochemical properties of the NLS-PE mutein in cancer cell models.
- To investigate the mechanism of action and potential alternative functions of the modified toxin.
Main Methods:
- Recombinant expression and purification of PE and its muteins.
- Cytotoxicity assays using A549 and HepG2 cell lines.
- Biochemical characterization of the modified toxin's activity.
Main Results:
- A novel PE mutein with an NLS sequence was successfully designed and purified.
- The NLS-PE mutein exhibited significant cytotoxic activity in tested cancer cell lines.
- Furin cleavage was not essential for PE activity in these cell lines.
- Nuclear accumulation of PE led to increased toxicity, suggesting a novel mechanism possibly involving nuclease activity and DNA cleavage.
Conclusions:
- The NLS-PE mutein demonstrates enhanced selective toxicity against cancer cells.
- PE possesses a potential alternative intoxication mechanism involving nuclease activity and chromosomal DNA cleavage.
- Further research is needed to elucidate the molecular basis of this newly discovered nuclease activity.
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