Mesoscopic p53-rich clusters represent a new class of protein condensates.

The protein p53 is an important tumor suppressor, which transforms, after mutation, into a potent cancer promotor. Both mutant and wild-type p53 form amyloid fibrils, and fibrillization is considered one of the pathways of the mutants' oncogenicity. p53 incorporates structured domains, essential to its function, and extensive disordered regions. Here, we address the roles of the ordered (where the vast majority of oncogenic mutations localize) and disordered (implicated in aggregation and condensation of numerous other proteins) domains in p53 aggregation. We show that in the cytosol of model breast cancer cells, the mutant p53 R248Q reproducibly forms fluid aggregates with narrow size distribution centered at approximately 40 nm. Similar aggregates were observed in experiments with purified p53 R248Q, which identified the aggregates as mesoscopic protein-rich clusters, a unique protein condensate. Direct TEM imaging demonstrates that the mesoscopic clusters host and facilitate the nucleation of amyloid fibrils. We show that in solutions of stand-alone ordered domain of WT p53 clusters form and support fibril nucleation, whereas the disordered N-terminus domain forms common dense liquid and no fibrils. These results highlight two unique features of the mesoscopic protein-rich clusters: their role in amyloid fibrillization that may have implications for the oncogenicity of p53 mutants and the defining role of the ordered protein domains in their formation. In a broader context, these findings demonstrate that mutations in the DBD domain, which underlie the loss of cancer-protective transcription function, are also responsible for fibrillization and, thus, the gain of oncogenic function of p53 mutants.