Mechanism of action of p28, a first in class, non-HDM2 mediated peptide inhibitor of p53 ubiquitination
T. Yamada, K. Christov, T.K. Das Gupta and C.W. Beattie. Division of Surgical Oncology, Department of Surgery, University of Illinois College of Medicine, Chicago, IL 60612.
Background: Exposure of a wide variety of wild type and mutated p53 cancer cells to a 28 aa peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post translational increase in the level of p53 and inhibits the cell cycle at G2/M. We now report an underlying mechanism for the increase in p53.
Methods: p53 wt MCF-7 and MCF-7 derived MDD2 p53dom/neg breast cancer cells were exposed to increasing doses of p28 for 24-72 hrs and Western analysis used to determine the levels of p53, the ubiquitin ligases HDM2, COP1, TOPORS and Pirh2, cyclin-dependent kinase inhibitor p21, G2/M-specific protein FoxM1, and stathmin (STMN1), an important regulatory protein of microtubule dynamics. We also used computer simulation (ClusPro, GROMACS) and domain specific antibodies to p53 to identify a potential site of p28 binding to p53.
Results: p28 significantly increased the levels of p53 and p21 in MCF-7 cells, while significantly reducing the level of FoxM1 over a 72 hr exposure. The level of STMN1 was not altered, suggesting the block at G2/M does not involve microtubules. p28 also decreased the level of COP1 > 80%, but did not significantly decrease TOPORS, Pirh2 or HDM2 levels in MCF-7 cells. In contrast, the levels of p53, p21, FoxM1, STMN1, COP1, TOPORS and Pirh2 either remained essentially at control levels (p53, p21) or were elevated (FoxM1, STMN1, COP1, TOPORS, Pirh2) at some point during a 72 hr exposure of p53dom/neg MDD2 cells to p28. Modeling and antibody studies suggest p28 binds to motifs that span aa 110-146 in flexible L1 loop and 80 and 276 of the p53 DNA binding domain (DBD), respectively. These data suggest that COP1, but not TOPORS or Pirh2, also binds within this region of p53 and that COP1 is a major regulator of p53 activity in breast cancer.
Conclusion: p28 binds to a specific motif within the DBD of p53 that blocks COP1 mediated degradation suggesting it is a prototype non-HDM2 mediated peptide inhibitor of p53 degradation.