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. 2021 Mar 9;118(10):e2015618118.
doi: 10.1073/pnas.2015618118.

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

David S Yang  1 , Arash Saeedi  1 , Aram Davtyan  2 , Mohsen Fathi  1 , Michael B Sherman  3   4 , Mohammad S Safari  1   5 , Alena Klindziuk  2 , Michelle C Barton  6 , Navin Varadarajan  1 , Anatoly B Kolomeisky  2   7 , Peter G Vekilov  8   9
Affiliations

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

David S Yang et al. Proc Natl Acad Sci U S A. .

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.

Keywords: fibrillization; nucleation mechanism; precursors.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
The R248Q mutation and aggregation on p53 R248Q in breast cancer cells. (A) The structure of the DBD (94 to 292) of wild-type p53 and the R248Q (Arg-248 Gln) mutant. Nitrogen atoms are colored in red, zinc in green, alpha helices in purple, beta sheets in orange, and DNA in blue. Protein Data Bank ID is 1TUP (23) for wild-type p53; prediction of p53 R248Q DNA interaction was done using Visual Molecular Dynamics (24). (B) Schematic of confocal immunofluorescence microscopy in which antibodies that specifically target cell components of interest are tagged with fluorescent dyes. The spatial distribution of the fluorescent signal collected by the microscope maps the 3D distribution of the target molecule. (C) Illustration of diffuse and punctate staining in the cytoplasm. (D) Combined staining of HCC70 cells with a Hoechst dye, which stains the nucleus, Pab240, which binds to unfolded or aggregated p53, and ThT, which recognizes amyloid structures. (E) Staining of HCC70 cells with Pab240 before and after treatment with 1,6-hexanediol, known to disperse dense liquid droplets of disordered proteins. (F) Distributions of the volumes of the puncta found in HCC70 cells treated with Pab240. Each trace represents the volume distribution of puncta from a single cell.
Fig. 2.
Fig. 2.
Mesoscopic protein-rich clusters of p53 R248Q. (A) Schematic of OIM. A 500-μm-thick solution layer is illuminated by a green laser (wavelength 532 nm) at an oblique angle. Upward scattered light is collected by a microscope lens. (B) Representative OIM micrographs tracing the evolution of aggregates in a 2-μM p53 R248Q solution at 15 °C. The observed volume is 5 × 80 × 120 μm3 depth × height × width. The clusters appear as gold speckles. (C) Number density distribution of the radii R of clusters determined by OIM at 2 μM and 15 °C. The averages of five measurements are displayed. The error bars represent the respective SDs. (D and E) The evolution of the average radius R and number N of clusters per unit solution volume in a 2-μM solution determined at 15 °C by OIM from images as in B. The averages of five measurements are displayed. The respective SDs are smaller than the symbol size. Horizontal lines denote the mean values of R and N. (F and G) The concentration dependence of R and N determined 15 °C by OIM. The averages of five measurements are displayed. The error bars represent the respective SDs and are smaller than the symbol size for most determinations. Horizontal line in F denotes the mean value of R; curve in G is a guide to the eye. (H and I) The concentration dependence of R and the cluster volume fraction ϕ2 determined at 15 °C by dynamic light scattering. The averages of five measurements are displayed. The error bars represent the respective SDs and are smaller than the symbol size for some determinations. Horizontal line in H denotes the mean value of R; the line in I is a guide to the eye. (J) Concentrations Cf and C2, defined in the plot after incubation for 20 min at 15 °C as a function of the initial solution concentration C0. (Inset) The Cf(C0) correlation in semilogarithmic coordinates. (K) Schematic of formation of mesoscopic p53-rich clusters owing to accumulation of transient misassembled oligomers, tentatively represented as pentamers and highlighted in ovals.
Fig. 3.
Fig. 3.
Fibrillization of wild type and p53 R248Q. (A–E) Negative staining EM micrographs reveal clusters, fibrils, and amorphous agglomerates of p53 R248Q in A–C and wild-type p53 in D and E. Gold arrows point to empty clusters; red arrows, clusters that spawn fibrils; and blue arrows, fibrils. (B) Branched fibrils coated with amorphous agglomerates. (C) Stand-alone amorphous agglomerates. (F–I) Evolution of the intensity of fluorescence at wavelength 500 nm of ANS in the presence of p53 at 37 °C. ANS concentrations were 200 µM in all tests. (F and G) At the listed concentrations of R248Q, in F, and wild type, in G, in the absence of Ficoll. (H and I) At 6.5 μM of R248Q, in H, and wild type, in I, and in the presence of varying concentrations of Ficoll. (J) Schematic of two-step nucleation of fibrils; step 1) Mesoscopic p53-rich clusters, comprised of misassembled p53 oligomers (depicted here as pentamers), native tetramers, and additional p53 species, assemble and step 2) Fibrils, which likely represent stacks of refolded p53 monomers (7), nucleate within the mesoscopic clusters by the assembly of p53 monomers. Fibril growth proceeds classically, via sequential association of p53 monomers from the solution. Compare to EM micrograph in A.
Fig. 4.
Fig. 4.
The free energy F of the conformations of wild type and p53 R248Q DBD. (A) As a function of the reaction coordinate q, which measures the similarity of the DBD conformation to a structure determined by X-ray crystallography (23). (B–E) 2D profiles of F as function of q and radius of gyration, Rg, in B and C, and end-to-end distance, D, in D and E, of the DBD chain. Gold arrows labeled with letters A through C point to the states of the structures in the respective panels in Fig. 5. The silver arrow labeled with a star indicates the state of the reference structure depicted with silver ribbons in Fig. 5 A–D.
Fig. 5.
Fig. 5.
Conformational changes induced by the R248Q mutation. (A) Comparison of wild-type DBD structures corresponding to F(q) minima at q = 0.555 (silver) and q = 0.515 (gold). Red box highlights the aggregation prone sequence, protected by the N terminus tail in wild-type p53 and exposed in p53 R248Q. Green star indicates residues 168 to 193, the location of the strongest deviation of between the two modeled wild-type conformations. (B–D) Comparisons of wild-type DBD structures corresponding to the F(q) minimum at q = 0.555 (silver) to the DBD structures of p53 R248Q (gold) at F(q) minima at q = 0.425 in B; at q = 0.445 in C; and at q = 0.455 in D. In A–D, the N terminus tail of the reference structure is highlighted in charcoal and that of the second structure in copper. (E) Free energy profiles F(q) for the cores of the DBDs (residues 107 to 276) of wild type and p53 R248Q.
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