Modulation of protein-protein interactions in the p53 pathway

Protein Protein Interactions (PPIs) are of remarkable significance given their role in the vast majority of cellular processes. The protein p53 is known as “the guardian of the genome” due to its involvement in DNA repair, inhibition of cell proliferation and cell cycle regulation in general. The activation of p53 is triggered by various stress signals, inducing a negative feedback loop that mediates levels of p53. The structurally similar proteins, hDMX and hDM2 bind to a transcriptional domain of p53, inhibiting its function in cell cycle repair or apoptosis. p53 is additionally degraded by hDM2 mediated ubiquitination, which is amplified by formation of hDMX/hDM2 heterodimers. Dimeric hub 14 3 3 proteins have a positive feedback effect on p53 activity and prior cellular studies have shown both hDMX and hDM2 undergo phosphorylation, which is recognized by these phospho binding 14 3 3 proteins. Stabilizing or inhibiting interactions of hDMX and hDM2 with 14 3 3 proteins might promote increased or decreased levels of cellular p53 respectively. Firstly, to fully characterize these interactions, a peptide based approach employing biophysical techniques (including FA, ITC and SPR) was used to determine the binding affinity between hDMX/hDM2 and 14 3 3 proteins. In addition, X ray crystallography was used to obtain structural information on the hDMX/14 3 3σ and hDM2/14 3 3σ interfaces. Finally, small molecule and fragment screening along with a protein templated fragment ligation approach was used to find novel small molecules that could modulate this pathway. This work provides better understanding of the role that 14 3 3 proteins and hDMX/hDM2 play in the p53 pathway, shining light on a novel approach to modulate PPIs in the p53 pathway.