Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo

The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin.

When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity.

These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.