The application of pressure has opened important frontiers for understanding how polypeptides fold into highly structured conformations, how they interact with ligands and other proteins, and how they assemble into supramolecular structures such as viruses and amyloids (1). Protein aggregation results in devastating neurodegenerative diseases and cancer. Our group has used high pressure to investigate the aggregation of amyloidogenic proteins (1, 2). In the case of the intrinsically disordered protein (IDP) α-synuclein, we used high hydrostatic pressure to identify the mechanism through which α-syn amyloid fibrils are dissociated into monomers (3, 4). We provide molecular evidence of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. We investigated the structural and dynamic properties of these monomers dissociated from HHP-disturbed fibrils and the remaining fibrillar species at the atomic level. We also examined how these species might seed amyloid fibril formation (4). We are now examining the disassembly profile of disease-related mutants of α-syn to evaluate the potential use of intermediates as targets for drug development. In the case of p53, its function is lost in more than 50% of human cancers. Studies from our laboratory and others have demonstrated that the formation of prion-like aggregates of mutant p53 is associated with loss-of-function, dominant-negative and gain-of-function (GoF) effects (5, 6, 7). p53 aggregates in a mixture of oligomers and fibrils that sequestrates the native protein into an inactive conformation. These aggregates are present in tissue biopsies of breast cancer especially in more aggressive ones. Perturbation of the p53 core domain (p53C) with sub-denaturing 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 (8,). We found that MG conformers of p53C, likely representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography (8). We also found that they bind the fluorescent dye bis-ANS and have a native-like tertiary structure as determined by NMR. 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 câncer (8). (This work was supported by CNPq, FAPERJ, FINEP and CAPES)

References:

[1] Silva JL et al. (2014). Chem Rev. 114: 7239-7267.

[2] Silva JL et al. (2010) Acc Chem Res, 43: 271-279.

[3] Foguel D et al. (2003) Proc. Natl. Acad. Sci. USA , 100: 9831-9836.

[4] de Oliveira GAP et al. (2016) Sci Rep, 2016 Nov 30; 6: 37990.

[5] Ishimaru D et al. (2003). Biochemistry, 42, 9022-9027.

[6] Ano Bom APD et al. (2012). J Biol Chem, 287: 28152-28162.

[7] Silva JL et al. (2014). Trends Biochem Sci. 39(6): 260-267.

[8] Silva JL et al. (2018). Acc Chem Res. 2018 Jan16;51(1):181-190.