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.