Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide.
Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.
CPBS, UMR 5236 CNRS, 1919 route de Mende, 34293 Montpellier, France.
Jocelyn
J
Méré
Christophe
C
Chopard
Anne
A
Bonhoure
Juliette
J
Morlon-Guyot
Bruno
B
Beaumelle
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D045683:Furin / Q000378:metabolism; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy*; D055550:Protein Stability; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
D000970:Antineoplastic Agents; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D007611:Aprotinin; D045683:Furin