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

Abstract

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 p53wt and p53mut 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 p53wt and p53mut 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.

A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

“…4,7,31,32 Configurations for azurin and the DBD of p63 were taken from 1E5Z and 3QYN of Protein Data Bank (PDB) files, respectively. ClusPro was used to conduct automated docking between the two molecules.…”

“…4,8 Human breast and melanoma cells were cultured with p28 at 50 µM for 24-72 hours, whole-cell lysates prepared, and Western analysis conducted. Polyclonal antibodies against p63 (H129; Santa Cruz Biotechnology), Itch (ab63940; Abcam, Cambridge, MA, USA), AIP5 (H260; Santa Cruz Biotechnology) and RACK-1 (ab62735; Abcam), and monoclonal p73 antibody (ER13; EMD Millipore) were used for Western analyses according to the suppliers' instructions.…”

“…4,5 Once bound to either wild type or mutated p53, 6,7 p28 blocks the binding of the E3 ubiquitin ligase Cop1 and induces its autodegradation. This increases the level and activity of p53 and the expression of its downstream cell cycle targets p21 and p27 significantly, inhibiting the cancer cell cycle at G 2 /M.…”

“…This increases the level and activity of p53 and the expression of its downstream cell cycle targets p21 and p27 significantly, inhibiting the cancer cell cycle at G 2 /M. 4,5,8 The structural similarity between p53 DBD and that of its homologues p63 and p73 suggests that p28 could also bind to either p63 or p73 and potentially enhance the reported tumor suppressive activity of individual isomers 4,7,9 through a similar mechanism of post-translational stabilization that alters their ubiquitination. 23 In addition to autoregulatory feedback loops, polyubiquitination regulates the stability of the p53 family of proteins through a series of E3 ubiquitin ligases divided into three subfamilies: the Homologous to E6-AP Carboxyl Terminus (HECT) domain-containing E3s, the Really Interesting New Gene (RING) finger domain-containing E3s, and the U box E3s.…”

“…5 gsT immunoprecipitation assays p28 binding to p53, p63, and p73 was analyzed using GST immunoprecipitation (pull-down) assays essentially as described. 4,5 GST and GST-p28 were bound to Glutathione Sepharose 4B beads (GE Healthcare) and unbound protein removed by washing with PBS. Whole-cell lysates of MCF-7, MDA-MB-231, Mel-29, -23, or -6 cells were mixed with beads and incubated for 2 hours.…”