Oncogenic p53 triggers amyloid aggregation of p63 and p73 liquid droplets

The tumor suppressor p53 is mutated in over half of human cancers, with mutants known to misfold and form amyloid aggregates that correlate with oncogenic gain-of-function and therapy resistance. While paralogs p63 and p73 share structural homology in their DNA-binding domains and are key tumor suppressors with elevated expression in some tumors, they display reduced aggregation propensity compared with p53. Prior work indicates mutant p53 can co-aggregate with p63/p73, impairing their function, but the mechanisms and species involved are unclear. Given growing evidence that liquid–liquid phase separation (LLPS) can precede amyloid formation in p53 and other nuclear proteins, the study asks whether p53 variants (WT and cancer mutants M237I, R248Q, R249S) can drive phase transitions of p63/p73 from liquid-like droplets to amyloid-like solids, and whether this process can be inhibited.

The paper situates the work within a body of research showing: (1) mutant p53 forms amyloid-like aggregates in diverse tumors (breast, skin, prostate, ovarian) and can seed aggregation of WT p53 and paralogs (prion-like behavior); (2) p63/p73 aggregate less than p53 due to differences in backbone hydrogen bond exposure and stability in their core domains; (3) LLPS is common among transcription factors and DNA repair proteins, and p53 LLPS can precede aggregation; (4) nucleic acids and polyanions modulate p53 folding, LLPS, and aggregation; (5) certain p53 mutations (e.g., M237I near Zn2+-binding site; R248Q/R249S DNA-contact/nearby) differentially affect stability and aggregation; and (6) inhibitors of p53 aggregation (e.g., heparin, small molecules like ADH-6, PRIMA-1, resveratrol) can mitigate amyloid formation. These findings motivate probing heterotypic LLPS/aggregation among p53 family members and testing inhibition strategies.

• Protein constructs: DNA-binding domains p53C (WT; mutants M237I, R248Q, R249S), p63C, p73C; full-length p53 WT and mutant M237I for cellular assays. Proteins expressed in E. coli and purified via affinity and heparin chromatography; labeled with NHS-Rhodamine (p63C/p73C) or Alexa Fluor 488 (p53C variants) for microscopy.
• Phase separation and aggregation assays: LLPS induced with PEG-4000 (typically 15% w/v). Microscopy (DIC/confocal) performed at 4, 25, and 37 °C across protein concentrations (10–100 µM). Temperature-shift experiments assessed droplet formation and conversion to aggregates. Congo red (CR, 10 µM) binding imaged for amyloid detection.
• Quantitative biophysics: Light scattering (excitation 320 nm; area under emission 300–340 nm) and turbidity (A600) measured to assess aggregation with/without PEG at 4/25/37 °C. Thioflavin T (ThT, 10 µM; ex 450 nm, em 460–500 nm, intensity at 470 nm) used to quantify amyloid formation.
• Seeding (prion-like conversion): p53C WT or M237I pre-aggregated at 37 °C for 3 min at 60 µM, then diluted 40-fold to 1.5 µM seeds and mixed with p63C (60 µM) under LLPS conditions (15% PEG). Aggregation monitored by LS/ThT and confocal microscopy tracking rhodamine-p63C, ThT positivity, and amyloid intrinsic visible fluorescence.
• Dual-color co-LLPS/co-aggregation: AF488-labeled p53C (WT/M237I/R248Q/R249S) mixed with rhodamine-labeled p63C or p73C (typically 40 µM each) at 4 and 37 °C to visualize co-condensation and co-aggregation.
• Heparin inhibition: Heparin (10 µM) added during seeding/co-aggregation assays to test inhibition of p53C-seeded conversion of p63C/p73C droplets.
• Circular dichroism: Far-UV CD (200–260 nm) of p63C alone and after co-aggregation with p53C WT or M237I to estimate secondary structure changes (SELCON3 deconvolution).
• Cell experiments: H1299 (p53-null) carcinoma cells cultured and transfected with full-length p53 M237I-EGFP alone or co-transfected with p63C-mCherry or p73C-YFP. Fixed-cell immunofluorescence for endogenous p73 colocalization; live-cell imaging to assess nuclear/nucleolar condensates. FRAP performed to quantify dynamics (nuclear vs nucleolar recovery).

• LLPS vs aggregation in vitro: p63C and p73C undergo LLPS in PEG at 4 °C across tested concentrations but do not intrinsically convert to amyloids at 25–37 °C. In contrast, p53C (WT and M237I) forms droplets at 4 °C and transitions to aggregates at 25–37 °C.
• Amyloid signatures: p53C and M237I aggregates bind Congo red and ThT strongly, whereas p63C/p73C droplets do not. Turbidimetry and LS corroborate aggregation in p53C but not in p63C/p73C under matching conditions.
• Quantitative differences: In 15% PEG at 25 °C, LS signals for p53C and M237I are ~10-fold and ~20-fold higher than p63C/p73C, respectively; at 37 °C, ~15-fold (p53C) and ~40-fold (M237I) higher. ThT binding increases relative to p63C/p73C by ~12-fold (p53C) and ~65-fold (M237I) at 25 °C, and ~22-fold (p53C) and ~120-fold (M237I) at 37 °C.
• Co-aggregation and seeding: Mixed samples show that p53C (WT and mutants) co-condense with p63C/p73C at 4 °C, and at 37 °C promote co-aggregation. Preformed p53C seeds (1.5 µM) convert non-amyloid p63C droplets (60 µM) into amyloid-like aggregates positive for ThT and intrinsic visible-range fluorescence; M237I exhibits stronger seeding than WT.
• Hotspot mutants: R249S and R248Q p53C seeds also convert p63C droplets to aggregates; R249S shows greater conversion than R248Q under tested conditions. Aggregate morphology differs among mutants (e.g., R273H forms more diffuse aggregates and shows weaker conversion than M237I/R249S).
• Secondary structure changes: CD shows co-aggregation with p53C WT/M237I drives p63C toward increased β-sheet content (e.g., with M237I in PEG at 37 °C, p63C α-helix ~2.7%, β-sheet ~41.3% vs ~11% and ~33.2% in solution).
• Cellular colocalization and dynamics: In H1299 cells, full-length p53 M237I-EGFP colocalizes with endogenous p73 in nucleolar condensates. Co-transfection with p63C-mCherry or p73C-YFP shows nuclear/nucleolar droplets with reduced FRAP recovery in nucleoli, indicating gel/solid-like transitions (approximate FRAP recovery: nuclear ~87% for p63C and ~77% for p73C; nucleolar ~42% for p63C and ~53% for p73C).
• Inhibition by heparin: Heparin (10 µM) blocks p53C (WT/M237I) seed-induced conversion of p63C and p73C droplets into aggregates, reducing LS signals while leaving droplets largely unaffected, demonstrating inhibition of prion-like templating.
• Sequence/LLPS propensity analyses: Bioinformatic predictions (FuzPred/FuzDrop) identify droplet-promoting and disorder-to-disorder regions across p53/p63/p73, including contributions from transactivation and SAM domains (in p63/p73) to LLPS; conserved hydrophobic segments within DNA-binding domains may influence phase behavior but do not fully explain p53’s higher amyloid propensity.

The study demonstrates that phase separation is a key step in the pathological aggregation of p53 and that p53 (both WT and mutants) can template amyloid-like conversion of p63 and p73 from their droplet states. This reveals a heterotypic prion-like mechanism within the p53 family that can impair paralog tumor-suppressive functions, potentially contributing to mutant p53 gain-of-function in cancer. Distinct p53 mutations exhibit different stabilities and seeding capabilities, yet multiple mutants (M237I, R249S, R248Q) can drive conversion, highlighting intrinsic aggregation-prone features centered on the p53 core domain. The nuclear environment, particularly nucleolar compartments rich in nucleic acids, appears to modulate condensate maturation and solidification; cellular FRAP data indicate stiffer, less dynamic nucleolar condensates consistent with amyloid-like transitions of p63C/p73C induced by mutant p53. Therapeutically, the successful use of heparin to block seeding-induced conversion underscores electrostatic modulation as a viable strategy to prevent aberrant phase transitions and amyloid formation. The findings align with broader models wherein LLPS can be on-pathway to aggregate formation for disease-linked proteins and position p53-driven heterotypic condensates as potential hubs for oncogenic dysregulation and drug targeting.

This work identifies a mechanistic link by which oncogenic p53 (WT or mutants) triggers amyloid aggregation of p63 and p73 by converting their liquid-like droplets into solid, amyloid-like assemblies. It establishes that p53C seeds are sufficient to drive this prion-like conversion in vitro and that full-length mutant p53 co-condenses with p63C/p73C in cells, where nucleolar condensates exhibit reduced dynamics indicative of solidification. Heparin halts seeding-induced aggregation, providing proof-of-principle for therapeutic inhibition of aberrant phase transitions. The study advances understanding of p53 family crosstalk in cancer and suggests targeting condensate maturation/aggregation as a strategy to restore tumor suppressor functions. Future research should develop selective inhibitors (e.g., polyanions, small molecules) of p53-driven heterotypic aggregation, dissect nucleic acid involvement, evaluate post-translational modification effects, and validate mechanisms in vivo.

• Mechanistic details of tripartite interactions among DNA/RNA, p53, and p63/p73 within the nuclear milieu remain unresolved; the specific nucleic acid sequences/stoichiometries that modulate conversion were not defined. - Cellular data were primarily from p53-null H1299 cells with overexpressed constructs, which may not fully recapitulate endogenous expression levels or tumor heterogeneity. - In vivo validation of the prion-like conversion mechanism and its impact on tumor suppression/oncogenesis was not performed. - The study focused on selected p53 mutants; broader mutant coverage and isoforms of p63/p73 were not exhaustively tested. - Heparin was used as a model polyanion inhibitor; its cellular uptake, specificity, and therapeutic practicality warrant further investigation, as do alternative drug-like inhibitors.