Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Abstract

The oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms that govern phase behaviors of the mutant across multiple length scales, from cellular to molecular. We establish that p53 R248Q forms mesoscopic proteinrich clusters, an anomalous liquid phase with several unique properties. We demonstrate that the clusters host and facilitate the nucleation of amyloid fibrils. The distinct characteristics of the clusters of R248Q and wild-type p53 and theoretical models indicate that p53 condensation into clusters is driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. Two-step nucleation of mutant p53 amyloids suggests means to IntroductionThe wild type p53 protein is a potent cancer suppressor, which is inactivated in almost every tumor, either through mutations in the TP53 gene (in 50% or more of human cancers) or deregulation of its associated pathways. 1-6 By contrast, p53 mutants emerge as potent cancer promoters because they exert a dominant-negative (DN) effect on the wild-type variant and also display oncogenic gain-of-function (GOF) properties by inhibiting other cancer suppressors. 1 Several mechanisms of cancer promotion by mutant p53 have been discussed. [6][7][8][9][10][11][12] It was recently suggested that the structural destabilization of the p53 mutants and the associated aggregation into insoluble, degradation resistant structures, designated fibrils, may play a decisive role in their oncogenicity. 2,[13][14][15] Concurrently, fibril suppression, for instance, by stabilization of the mutant p53 conformation, has been identified as a general way to fight cancer. 2,16 A parallel development identified a new form of biological organization, liquid-liquid phase separation, which constitutes several common membraneless organelles such as nucleoli, Cajal bodies, and P granules. [17][18][19][20][21] Many proteins forming dense liquid condensates in live cells incorporate significant disordered regions and condensation at the low concentrations of these proteins in the nucleoplasm and cytoplasm has been attributed to stronger intermolecular attraction due to the unstructured segments. 19,20 Furthermore, it is well-appreciated that protein dense liquids can serve as precursors and facilitators of higher order organization such as ordering into crystals 22-25 and the formation of polymers of sickle hemoglobin. 26,27 Theoretical analyses and recent experiments suggest that a similar precursor mechanism would apply to the nucleation of amyloid fibrils. [28][29][30][31][32][33]

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

“…Remarkably, at 37°C, R increases to ca. 150 nm (34), which is consistent with the average volume of the intracellular clusters in HCC70 cells (Fig. 1F).…”

Biomolecular Tau condensation is linked to Tau accumulation at the nuclear envelope

“…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, 2020).…”

Two-Step Crystal Nucleation Is Selected Because of a Lower Surface Free Energy Barrier

“…As a consequence, OIM measures sizes in the range from 20 nm to 5 μm, much smaller than the diffraction limit of conventional optical microscopy. 23,[57][58][59]66 OIM revealed insulin aggregates in both the acetone-free and acetone-containing solutions (Figure 6a,b) 67 The average aggregate size, between 160 and 200 nm, appears to be insensitive to protein concentration and somewhat larger in the presence of acetone than in acetonefree solutions (Figure 6c,d). Aggregates of this size would hold about 100 000 insulin hexamers in loose contact.…”