Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants.
Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.
Department of Chemistry Paolo Corradini, University of Naples Federico II, Italy. concetta.giancola@unina.it
Concetta
C
Giancola
Carmine
C
Ercole
Iolanda
I
Fotticchia
Roberta
R
Spadaccini
Elio
E
Pizzo
Giuseppe
G
D'Alessio
Delia
D
Picone
D000818:Animals; D041702:BALB 3T3 Cells; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004722:Endoribonucleases; C032523:ribonuclease SPL