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Anthrax Toxin-Mediated Delivery of the Pseudomonas Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions
- Source :
- mBio, mBio, Vol 4, Iss 3 (2013)
- Publication Year :
- 2013
- Publisher :
- American Society for Microbiology, 2013.
-
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.<br />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.
- Subjects :
- Virulence Factors
Recombinant Fusion Proteins
Anthrax toxin
Bacterial Toxins
Exotoxins
Antineoplastic Agents
Ubiquitin-conjugating enzyme
Microbiology
Deubiquitinating enzyme
Mice
Cytosol
Ubiquitin
Cell Line, Tumor
Neoplasms
Virology
Animals
Pseudomonas exotoxin
ADP Ribose Transferases
Antigens, Bacterial
Biological Products
biology
Fusion protein
QR1-502
Ubiquitin ligase
Mice, Inbred C57BL
Disease Models, Animal
Ubiquitins
Treatment Outcome
Biochemistry
Proteolysis
biology.protein
Female
Carrier Proteins
Research Article
Subjects
Details
- ISSN :
- 21507511 and 21612129
- Volume :
- 4
- Database :
- OpenAIRE
- Journal :
- mBio
- Accession number :
- edsair.doi.dedup.....daa29b509349de5a9696df57ca35b5d5