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Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases

Laboratori d’Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, Maria Aurèlia Capmany 40, 17003 Girona, Spain
Institut d’Investigació Biomèdica de Girona Josep Trueta, (IdIBGi), 17190 Girona, Spain
Author to whom correspondence should be addressed.
Molecules 2018, 23(12), 3273;
Received: 8 November 2018 / Revised: 5 December 2018 / Accepted: 8 December 2018 / Published: 11 December 2018
(This article belongs to the Section Chemical Biology)
Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered. View Full-Text
Keywords: antitumor protein; nuclear-directed ribonuclease; disulfide bond; thermal stability; proteolytic resistance antitumor protein; nuclear-directed ribonuclease; disulfide bond; thermal stability; proteolytic resistance
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MDPI and ACS Style

Roura Padrosa, D.; Castro, J.; Romero-Casañas, A.; Ribó, M.; Vilanova, M.; Benito, A. Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases. Molecules 2018, 23, 3273.

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