Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer

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.

The Status of p53 Oligomeric and Aggregation States in Cancer

“…The numerous studies on p53 DBD aggregation suggest that it follows the general model of amyloid formation: monomer → misfolded monomer → dimer → oligomer → protofibril → mature fibril (Figure 4). Under sub-denaturing concentrations of guanidine hydrochloride, the structural ensemble of the p53 DBD can be shifted to populate swollen molten globule states with native like secondary and tertiary structure, and accelerated aggregation kinetics [78]. An open question is to define the oligomeric/multimeric species of mutant p53 or altered p53 that exert GoF activity resulting in higher malignancy.…”

“…Structurally destabilizing p53 DBD mutations can result in a similar outcome [43]. As such, native-state stabilization is a primary avenue being explored to impede p53 DBD aggregation [23,78]. Most approaches to date endeavor to use small molecules to stabilize particular mutants, with the majority of efforts focusing on the cavity induced by the Y220C hotspot mutation [79].…”

Tetramer formation by the caspase‐activated fragment of the Par‐4 tumor suppressor

“…This suggests the low‐salt conformation may resemble an intermediate in the folding process. Interestingly, p53 MG conformers are highly prone to aggregation and amyloid‐like oligomers . The same could occur with cl‐Par‐4 MG conformers.…”

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

“…For example, prion-forming sequences, which are especially enriched in asparagine, have been shown to promote molten globule-like structures, in which amyloid-nucleating contacts can be made [55,56]. Mutant forms of the tumor suppressor protein, p53, have recently been shown to behave as molten globules while exhibiting prion-like properties and, although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression [57,58].…”