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Summary Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer., Graphical Abstract, Highlights • Amyloid oligomers transform p53 tumor suppressor into an oncogene • Amyloid-like mutant p53 oligomers occur in chemoresistant glioblastoma cells • p53 oligomer larger than tetramers is detected in the nuclei of living cells • Gain-of-function p53 phenotypes is attributed to p53 amyloid oligomers, Structural Biology; Protein Structure Aspects; Biophysics; Cancer

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

Since Bridgman's seminal experiments on high-pressure denaturation of albumen in 19141, the origin of pressure unfolding of proteins has remained unresolved. We report here a systematic study of the contribution of cavities to the volume difference between unfolded and folded states (ΔVu), using 10 single point variants of staphylococcus nuclease (SNase). Each mutation is localised in a strategic position and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the SNase structure. For every variant, a crystal structure confirmed the presence of the designed cavity with no detectable presence of water molecules. High-pressure fluorescence experiments show significantly larger ΔVu values for the cavity mutants in comparison to the reference protein. This demonstrates that solvent-excluded cavities make a major contribution to ΔVu. Thus, pressure effects have their origin in a property of the folded states of proteins, unlike temperature and chemical denaturants, whose effects are governed by exposed surface area in the unfolded state. High-pressure NMR experiments on 4 cavity mutants, recording HSQCs peak intensities up to 2500 bar, allowed precise estimations of the apparent ΔVu monitored by more than two-thirds of the residues. An innovative combination of the site-specific NMR data and Go-model simulations revealed significant departures from the apparent two-state folding process for the SNase reference protein and the cavity mutants. This study opens up highly promising perspectives on the use of high pressure for characterization of folding landscapes inaccessible by other methods.1. Bridgman, P. W. J. Biol. Chem. 1914.19, 511–512.