Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases.

Abstract

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.

• Authors: David Roura Padrosa, Jessica Castro, Alejandro Romero-Casañas, Marc Ribó, Maria Vilanova, Antoni Benito
• Journal: Molecules (Basel, Switzerland) (Molecules) Vol. 23 Issue 12 (Dec 11 2018) ISSN: 1420-3049 [Electronic] Switzerland
• PMID: 30544927 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents
• Disulfides
• Nuclear Localization Signals
• Ribonuclease, Pancreatic
• Endopeptidase K

Topics

• Antineoplastic Agents (pharmacology)
• Cell Line, Tumor
• Disulfides (chemistry)
• Endopeptidase K (chemistry, metabolism)
• Enzyme Stability
• Humans
• Mutagenesis, Site-Directed
• Nuclear Localization Signals (genetics)
• Protein Engineering (methods)
• Proteolysis
• Ribonuclease, Pancreatic (chemistry, genetics, metabolism, pharmacology)