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An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo
C A Midgley1, J M Desterro, M K Saville
1Department of Biochemistry, University of Dundee, Scotland, UK.
Abstract:
The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.
Insights
A novel peptide derived from p14ARF protein (Peptide 3) binds Mdm2, inhibiting p53 degradation. This peptide activates p53 transcription and may offer therapeutic potential for tumors with wild-type p53.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Degradation
Background:
- The p53 tumor suppressor is crucial for preventing cancer, but its activity is often inhibited by Mdm2.
- Mdm2 targets p53 for degradation via the ubiquitin-proteasome pathway.
- p14ARF is another tumor suppressor that inhibits Mdm2, thereby stabilizing p53.
Purpose of the Study:
- To identify specific p14ARF-derived peptides that can bind Mdm2.
- To investigate the potential of such peptides to activate p53 function.
- To explore the therapeutic implications of blocking p53 degradation.
Main Methods:
- Screening of synthetic peptides from p14ARF sequence.
- Deletion mapping to identify the Mdm2 binding site for Peptide 3.
- Expression of a green fluorescent protein-Peptide 3 fusion in cancer cell lines (MCF7, U-2 OS).
- In vitro ubiquitination assays to assess Mdm2's effect on p53.
Main Results:
- A peptide (Peptide 3) corresponding to the first 20 amino acids of ARF was found to bind human Mdm2.
- Peptide 3's binding site on Mdm2 was mapped adjacent to the 2A10 antibody binding site.
- Expression of Peptide 3 induced p53 protein and p53-dependent transcription in cancer cells.
- Peptide 3 inhibited Mdm2-mediated in vitro ubiquitination of p53.
Conclusions:
- Peptide 3 effectively binds Mdm2 and activates p53.
- This peptide can block Mdm2-induced p53 degradation.
- Small peptides targeting Mdm2-p53 interaction represent a potential therapeutic strategy for cancers with functional p53.