Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric ScFvs blocks the E6-mediated degradation of cellular p53.
The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.
Giovane
C
C
Ecole Supérieure de Biotechnologie de Strasbourg, ULP/UPRES 1329, boulevard Sébastien Brant, 67400 Illkirch, France.
Trave
G
G
Briones
A
A
Lutz
Y
Y
Wasylyk
B
B
Weiss
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D027383:Papillomaviridae / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012097:Repressor Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000473:pathology
D000911:Antibodies, Monoclonal; C068651:E6 protein, Human papillomavirus type 16; D000939:Epitopes; D007128:Immunoglobulin Fragments; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; C055966:immunoglobulin Fv