Nucleation and Condensation of Modified p53: a Baseline to Understand p53 Aggregation

Cancer is one of the leading causes of death worldwide. The protein p53 is an important tumor suppressor and is known as the guardian of the genome. This protein is a transcription factor that binds to DNA and controls multiple signaling pathway to determine the cell fate. More than 50 % of human cancers are related to mutations in the p53 DNA-binding domain. Recent studies suggest that p53 aggregation is a key factor in cancer development and the majority of the p53 mutants have an exaggerated propensity to aggregate. Mechanistic details on the nucleation and growth of p53 amyloid fibrils, however, are missing. We explore the aggregation mechanism of the p53 R248Q mutant by combining immunofluorescent 3D confocal microscopy of breast cancer with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms of phase behavior and aggregation. We establish that R248Q p53 forms anomalous condensates which host nucleation of amyloid fibrils. We also demonstrate that in contrast to dense liquids of other partially disordered proteins, the p53 clusters are driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. The proposed two-step aggregation pathway is supported by data on the aggregation of a protein construct in which we removed the disordered domains and left intact the ordered DNA binding domain of p53. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Furthermore, we also investigate the coaggregations of mutant p53 with wild type p53 and p63 to understand how two step nucleation can be related to oncogenic behavior of mutant p53. In a broader context, our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.