The possible use of ribonucleases in the treatment of diseases, including cancer, has received a considerable interest in the last years. The necessary requirements that convert a ribonuclease into a cytotoxin for malignant tumour cells include binding to cell membrane, translocation into the cytosol and RNA degradation. Among the mammalian ribonucleases, only bovine seminal ribonuclease (BS-RNase) fulfils all these requirements (1). We have designed a BS_RNase variant with an enforced cluster of positive charges at the N-termini surface to improve its cytotoxic potential according to literature data indicating that the protein approaches the membrane with a preferred orientation (2). We found that the G38K-BS-RNase variant displays an increased citotoxicity for tumour cells and we hypothesized the higher cytotoxicity to be connected to its enhanced interaction with membranes. A detailed investigation of the membrane interaction mechanism of BS-RNase and G38K-BS-RNase, using a combination of biophysical techniques, including Surface Plasmon Resonance and Electron Spin Resonance, supports this idea, and indicates for both of them a common two-step mechanism, based on an initial binding to the bilayer surface followed by a shallow penetration in the lipid core. Protein binding , driven by electrostatic interactions, effectively perturbs lipid packing and dynamics. Our results, while ruling out the hypothesis that the protein crosses the membrane interior, support the hypothesis of a BS-RNase entry mediated by an endocytosis mechanism. Overall, these studies shed light on the mechanism of ribonuclease membrane binding and perturbation, and provide a rational basis to design proteins with higher antitumor potential.

Design of more potent antitumor ribonucleases: a cluster of positive charges at N-termini enhances the cytotoxicity of bovine seminal ribonuclease via a membrane-mediated mechanism

PICONE, DELIA;D'ERRICO, GERARDINO;ERCOLE, CARMINE;GALDIERO, STEFANIA;
2011

Abstract

The possible use of ribonucleases in the treatment of diseases, including cancer, has received a considerable interest in the last years. The necessary requirements that convert a ribonuclease into a cytotoxin for malignant tumour cells include binding to cell membrane, translocation into the cytosol and RNA degradation. Among the mammalian ribonucleases, only bovine seminal ribonuclease (BS-RNase) fulfils all these requirements (1). We have designed a BS_RNase variant with an enforced cluster of positive charges at the N-termini surface to improve its cytotoxic potential according to literature data indicating that the protein approaches the membrane with a preferred orientation (2). We found that the G38K-BS-RNase variant displays an increased citotoxicity for tumour cells and we hypothesized the higher cytotoxicity to be connected to its enhanced interaction with membranes. A detailed investigation of the membrane interaction mechanism of BS-RNase and G38K-BS-RNase, using a combination of biophysical techniques, including Surface Plasmon Resonance and Electron Spin Resonance, supports this idea, and indicates for both of them a common two-step mechanism, based on an initial binding to the bilayer surface followed by a shallow penetration in the lipid core. Protein binding , driven by electrostatic interactions, effectively perturbs lipid packing and dynamics. Our results, while ruling out the hypothesis that the protein crosses the membrane interior, support the hypothesis of a BS-RNase entry mediated by an endocytosis mechanism. Overall, these studies shed light on the mechanism of ribonuclease membrane binding and perturbation, and provide a rational basis to design proteins with higher antitumor potential.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/451795
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