Abstract
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.
Highlights
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
Therapeutics directed at transient [17] and pre-amyloidogenic molten globule (MG)5 p53 conformers may be effective against some mutant classes [18]
Together with chemical (guanidine hydrochloride (GuHCl)) and physical approaches, we explored the energetic landscape of p53 core domain (p53C) and its homologs p63C and p73C and elucidated features specific to its pre-amyloidogenic states
Summary
Unlike the Trp in p63C and p73C, which localizes to the beginning of -strand S2, Trp-146 in p53C is located at the end of -strand S3 (Fig. 1). The result suggests that there are few changes in tertiary structure within the ensemble, providing additional evidence of a population of native-like MG states of p53C in 0.8 M GuHCl. At higher GuHCl concentrations (1.2 and 2 M), the NMR spectra revealed unfolding (Fig. 5, d and e) in agreement with fluorescence data (Fig. 2c). The volume changes of F 7 U (i.e. 0 M GuHCl), FЈ 7 U (i.e. 0.5 M GuHCl), and FЉ 7 U (i.e. 0.8 M GuHCl) ensembles were obtained from the transition points of the HHP titrations (see “Experimental procedures”) after plotting the raw fluorescence data (Fig. 4a) as unfolded fractions against the pressure values (Fig. 6a and Table 2).
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