title: Reduction of furin-nicked Pseudomonas exotoxin A: an unfolding story.
abstract: Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytically processed by furin to produce an N-terminal fragment of 28 kDa and a C-terminal fragment of 37 kDa. Cleavage is followed by the reduction of a key disulfide bond (cysteines 265-287). This combination of proteolysis and reduction releases the 37 kDa C-terminal fragment, which then translocates to the cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. To investigate toxin reduction, furin-nicked PE or a hypercleavable mutant, PEW281A, was subjected to various treatments and then analyzed for fragment production. Reduction was evident only when unfolding conditions and a reducing agent were applied. Thermal unfolding of PE, as evidenced by changes in alpha-helical content and increased sensitivity to trypsin, rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) mediated reduction. When subcellular fractions from toxin-sensitive cells were incubated with nicked PE, toxin unfolding and reducing activities were present in the membrane fraction but not the soluble fraction. These data indicate that PE reduction is a two-step process: unfolding that allows access to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulfur 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" conformation that restricts solvent access to the Cys265-287 disulfide bond until after a cell-mediated unfolding event.
authors.affiliation: Biotherapy Section, Laboratory of Molecular Biology, Division of Basic Science, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: McKee
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002458:Cell Fractionation; D006224:Cricetinae; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism; D045683:Furin; D006358:Hot Temperature; D006868:Hydrolysis; D007739:L Cells; D051379:Mice; D010084:Oxidation-Reduction; D017510:Protein Folding; D011550:Pseudomonas aeruginosa; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D045683:Furin; D013381:Subtilisins; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A