Inhibition of E6-induced degradation of its cellular substrates by novel blocking peptides.
The E6 oncoprotein derived from the tumour-associated human papillomavirus (HPV) types induces the ubiquitin-mediated degradation of several cellular proteins by conjugating them with the cellular ubiquitin ligase E6-AP. This is a HECT domain-containing ligase that was originally identified through its involvement in the E6-mediated degradation of the cellular tumour suppressor protein p53. Here we have investigated, in more detail, the nature of the E6/E6-AP interaction using binding peptides isolated from an E6-specific library. The selected peptides were either predicted or shown to have an alpha-helical core resembling the E6-binding motif on E6-AP, as well as amino acid alterations that increased their affinity for E6. These peptides were potent inhibitors of the E6/E6-AP interaction. Further analysis of the effects of these peptides on the ability of E6 to direct the proteolytic degradation of its various substrates, including p53, Dlg and the MAGI family of proteins, as well as using E6-AP immunodepletion, revealed striking differences in the mechanism by which E6 targets its cellular substrates for degradation. These results suggest that the site on E6 bound by E6-AP is also most likely occupied by other, as yet unidentified, ubiquitin ligases.
Sterlinko Grm
Helena
H
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
Weber
Malte
M
Elston
Rob
R
McIntosh
Pauline
P
Griffin
Heather
H
Banks
Lawrence
L
Doorbar
John
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051528:Guanylate Kinases; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D004791:Enzyme Inhibitors; D009856:Oncogene Proteins, Viral; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases