Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions

ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit...

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Autores principales:	Christopher Bachran, Thomas Morley, Suzanne Abdelazim, Rasem J. Fattah, Shihui Liu, Stephen H. Leppla
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bf0f3719e73844cda27ef4d099143852
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spelling	oai:doaj.org-article:bf0f3719e73844cda27ef4d0991438522021-11-15T15:40:05ZAnthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions10.1128/mBio.00201-132150-7511https://doaj.org/article/bf0f3719e73844cda27ef4d0991438522013-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00201-13https://doaj.org/toc/2150-7511ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. IMPORTANCE Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.Christopher BachranThomas MorleySuzanne AbdelazimRasem J. FattahShihui LiuStephen H. LepplaAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 3 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Christopher Bachran Thomas Morley Suzanne Abdelazim Rasem J. Fattah Shihui Liu Stephen H. Leppla Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
description	ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. IMPORTANCE Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.
format	article
author	Christopher Bachran Thomas Morley Suzanne Abdelazim Rasem J. Fattah Shihui Liu Stephen H. Leppla
author_facet	Christopher Bachran Thomas Morley Suzanne Abdelazim Rasem J. Fattah Shihui Liu Stephen H. Leppla
author_sort	Christopher Bachran
title	Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
title_short	Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
title_full	Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
title_fullStr	Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
title_full_unstemmed	Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
title_sort	anthrax toxin-mediated delivery of the <italic toggle="yes">pseudomonas</italic> exotoxin a enzymatic domain to the cytosol of tumor cells via cleavable ubiquitin fusions
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/bf0f3719e73844cda27ef4d099143852
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Anthrax Toxin-Mediated Delivery of the <italic toggle="yes">Pseudomonas</italic> Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions

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