title: Generation of new cytotoxic human ribonuclease variants directed to the nucleus.
abstract: Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5.
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, M. Aurélia Campmany 69, Girona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Vert
authors.forename: Jessica
authors.initials: J
authors.lastname: Castro
authors.forename: Santiago
authors.initials: S
authors.lastname: Ruiz-Martínez
authors.forename: Pere
authors.initials: P
authors.lastname: Tubert
authors.forename: Diego
authors.initials: D
authors.lastname: Escribano
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.forename: Antoni
authors.initials: A
authors.lastname: Benito
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation; D019913:Nuclear Localization Signals / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015138:RNA, Nuclear / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D019913:Nuclear Localization Signals; D015138:RNA, Nuclear; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic