RESUMO
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.
RESUMO
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.
Assuntos
Neoplasias/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Humanos , Modelos Moleculares , Agregados Proteicos , Conformação Proteica , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Estabilidade Proteica , Termodinâmica , Proteína Supressora de Tumor p53/químicaRESUMO
Virus dissociation and inactivation by high pressure have been extensively studied in recent decades. Pressure-induced dissociation of viral particles involves a reduction in the Gibbs free energy of dissociation and a negative change in volume. In this work, we investigated the combined effect of high pressure and temperature on the dissociation of tobacco mosaic virus (TMV). We assumed the presence of two states of TMV with different tendencies to dissociate. Thus one form presents a low tendency (L) and the other a high tendency (H) to dissociate. Based on the model described here, the L-H transition was favored by an increase in pressure and a decrease in temperature. The volume change of dissociation was pressure- and temperature-dependent, with a highly negative value of -80 mL/mol being recorded at 0 °C and atmospheric pressure. The entropy and enthalpy of dissociation were very temperature- and pressure-dependent, with values of entropy of 450 to -1300 kJ/mol and values of enthalpy of 5.5 × 10(4) to 2.4 × 10(4) kJ/mol. The dissociation of TMV was enthalpy-driven at all temperatures and pressures investigated. Based on these findings, we conclude that the model presented allows accurate predictions of viral dissociation behavior in different experimental conditions.