p28, A first in class peptide inhibitor of cop1 binding to p53

• Published in: British journal of cancer Vol. 108; no. 12; pp. 2495 - 2504
• Main Authors: Yamada, T, Christov, K, Shilkaitis, A, Bratescu, L, Green, A, Santini, S, Bizzarri, A R, Cannistraro, S, Gupta, T K D, Beattie, C W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25-06-2013; Nature Publishing Group
• Subjects: 631/67/581; 631/80; Amino Acid Sequence; Animals; Azurin - chemistry; Azurin - metabolism; Azurin - pharmacology; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Breast cancer; Cancer Research; Cell cycle; Cell growth; Cell Line, Tumor; Cluster analysis; Down-Regulation - drug effects; Drug Resistance; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Epidemiology; Female; Humans; Indexing in process; Male; Medical sciences; Melanoma; Mice; Mice, Nude; Models, Molecular; Molecular Dynamics Simulation; Molecular Medicine; Molecular Sequence Data; Oncology; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Peptides; Protein Binding - drug effects; Protein Interaction Domains and Motifs - drug effects; Protein Interaction Domains and Motifs - physiology; Proteins; Simulation; Translational Therapeutics; Tumor Suppressor Protein p53 - antagonists & inhibitors; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - metabolism; Tumors; Ubiquitin-Protein Ligases - antagonists & inhibitors; Ubiquitin-Protein Ligases - metabolism; Xenograft Model Antitumor Assays; United States > US; COP1; E3 ligases; cell cycle; ubiquitination; cell penetrating peptide; p53; Ubiquitination; Cancerology; TP53 Gene; Cell penetrating peptide; Enzyme; Ligases; Cell cycle; Inhibitor; Tumor suppressor gene
• ISSN: 0007-0920; 1532-1827
• DOI: 10.1038/bjc.2013.266

Background:
A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen
Pseudomonas aeruginosa,
produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination.
Methods:
In silico
computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding.
In vitro
direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions.
Results:
The L1 loop (aa 112–124), a region within the S7–S8 loop (aa 214–236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53
wt
and p53
mut
cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53
wt
and p53
mut
cells were in response to sustained increases in p53.
Conclusion:
These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.