Contribution of the C30/C75 disulfide bond to the biological properties of onconase.
Onconase, a member of the pancreatic type ribonuclease family, is currently used as a chemotherapeutic agent for the treatment of different types of cancer. It is widely accepted that one of the properties that renders this enzyme cytotoxic is its ability to evade the cytosolic ribonuclease inhibitor (RI). In the present work, we produced and characterized an onconase variant that lacks the disulfide bond C30/C75. This variant mimics the stable unfolding intermediate des(30-75) produced in the reductive unfolding pathway of onconase. We found that the reduction of the C30/C75 disulfide bond does not significantly alter the cytotoxic properties of onconase, although the variant possesses a notably reduced conformational stability. Interestingly, both its catalytic activity and its ability to evade RI are comparable to wild-type onconase under mild reductive conditions in which the three disulfide containing intermediate des(30-75) is present. These results suggest that the C30/C75 disulfide bond could easily be reduced under physiological redox conditions.
Torrent
Gerard
G
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n, E-17071 Girona, Spain.
Benito
Antoni
A
Castro
Jessica
J
Ribó
Marc
M
Vilanova
Maria
M
D001695:Biology; D002244:Carbon / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
D004220:Disulfides; D004791:Enzyme Inhibitors; D002244:Carbon; D012260:Ribonucleases; C058703:ranpirnase