Reduction of furin-nicked Pseudomonas exotoxin A: An unfolding story

Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytical ly processed by furin to produce an N-terminal fragment of 28 kDa and a C-t erminal fragment of 37 kDa. Cleavage is followed by the reduction of a key disulfide bond (cysteines 265-287). This combination of proteolysis and red uction releases the 37 kDa C-terminal fragment, which then translocates to the cytosol where it ADP-ribosylates elongation factor 2 and inhibits prote in synthesis. To investigate toxin reduction, furin-nicked PE or a hypercle avable mutant, PEW281A, was subjected to various treatments and then analyz ed for fragment production. Reduction was evident only when unfolding condi tions and a reducing agent were applied. Thermal unfolding of PE, as eviden ced by changes in or-helical content and increased sensitivity to trypsin, rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) me diated reduction. When subcellular fractions from toxin-sensitive cells wer e incubated with nicked PE, toxin unfolding and reducing activities were pr esent in the membrane fraction but not the soluble fraction. These data ind icate that PE reduction is a two-step process: unfolding that allows access to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulf ur bond by PDI or a PDI-like enzyme. With regard to cellular processing, we propose that the toxin's three-dimensional structure retains a "closed" co nformation that restricts solvent access to the Cys265-287 disulfide bond u ntil after a cell-mediated unfolding event.