Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils

Abstract

The protein p53 is a crucial tumor suppressor, often called “the guardian of the genome”; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates

“…They may thus play an important role for functional and pathological LLPS of Tau. Recently, similar mesoscopic clusters were reported for p53 in cells (Yang et al, 2021) and for RNA binding proteins in vitro (preprint: Kar et al, 2022).…”

14-3-3 Proteins are Potential Regulators of Liquid–Liquid Phase Separation

“…While experiments using recombinant p53 suggest that electrostatic interactions between the negatively charged N-terminal domain and the positively charged C-terminal domain play a key role in mediating p53 droplet formation [166], some mutations are found to markedly promote p53 condensation in cancer cells [167,168]. Though the function of p53 condensates remains elusive [169], formation of p53 droplets may be related to p53 aggregation [164,165,168,170].…”

Temperature Modulation of the DBDp53 Structure as Monitored by Static and Time-Resolved Fluorescence Combined with Molecular Dynamics Simulations

“…In this respect, we note some variability in the position of L 3 with respect to that of Arg248, which is known to play a crucial role in DNA binding. 52 Furthermore, some structural changes with temperature also appear in the region around the Trp146 residue.…”