Characterizing the role of p53 interactors in cell fate decisions

The tumour suppressor p53 is considered to be one of the most studied proteins of all time, and yet much remains to be discovered about this enigmatic protein. As a stress response protein, p53 is able to coordinate multiple signalling inputs to alter cell fate to either cause cell cycle arrest in the form of senescence or death via apoptosis. With regards to senescence, there is a paucity of data demonstrating how p53 elicits and maintains this phenotype. To overcome this gap, interactors of p53 in the context of senescence were identified using two main approaches, immunoprecipitation and proximity labelling of p53, in a well characterized cellular model of senescence. An IP at different times over the first 72 hours of senescence induction analysed by tandem mass spectrometry identified known interactors of p53, such as heat shock proteins and 14-3-3, but also potential novel interactors such as Synemin. Proximity labelling studies were next conducted by generating chimeric p53-APEX2 proteins at both the N and C terminal ends of p53 to identify further possible interactors. Following labelling with biotin, samples over 72 hours were similarly analysed by tandem mass spectrometry. This resulted in the identification of numerous proteins, local to p53 throughout the early stages of senescence and potentially implicated in various aspects of the senescent phenotype, such as the SASP, lysosome translocation, cytoskeletal organisation, and cell cycle arrest. Together, these proteins could be setting up a “pro-senescent” balance that would help determine p53-dependent cell fate decisions. This is the first dynamic interactome of p53 in the first stages of senescence, and it provides novel insights on how p53 mediates this phenotype. Among the known modulators of p53, we further studied the retinoic acid receptor, Stra6, which increased ROS and p53 levels and reduced cell viability when expressed in DNA damaged cells. Stra6 may play a role in cell fate decisions by enhancing apoptotic responses. This connection between p53 and the retinoic acid pathway warrants further attention to fully understand the mechanism at work.