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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

Herein, we report the synthesis and characterization of two dinuclear FeIIIZnII complexes [FeIIIZnIILP1] (1) and [FeIIIZnIILP2] (2), in which LP1 and LP2 are conjugated systems containing one and two pyrene groups, respectively, connected via the diamine -HN(CH2)4NH- spacer to the well-known N5O2-donor H2L ligand (H2L = 2-bis{[(2-pyridylmethyl)aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol). The complex [FeIIIZnIIL1] (3), in which H2L was modified to H2L1, with a carbonyl group attached to the terminal phenol group, was included in this study for comparison purposes.1 Both complexes 1 and 2 were satisfactorily characterized in the solid state and in solution. Extended X-ray absorption fine structure data for 1 and 3 in an acetonitrile solution show that the multiply bridged structure seen in the solid state of 3 is retained in solution. Potentiometric and UV-vis titration of 1 and 2 show that electrostatic interaction between the protonated amino groups and coordinated water molecules significantly decreases the pKa of the iron(III)-bound water compared to those of 3. On the other hand, catalytic activity studies using 1 and 2 in the hydrolysis of the activated substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) resulted in a significant increase in the association of the substrate (Kass ≅ 1/KM) compared to that of 3 because of electrostatic and hydrophobic interactions between BDNPP and the side-chain diaminopyrene of the ligands H2LP1 and H2LP2. In addition, the introduction of the pyrene motifs in 1 and 2 enhanced their activity toward DNA and as effective antitumor drugs, although the biochemical mechanism of the latter effect is currently under investigation. These complexes represent interesting examples of how to promote an increase in the activity of traditional artificial metal nucleases by introducing second-coordination-sphere effects.

Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance to standard treatments. Amyloid-like oligomeric species of mutant p53 have been attributed to transformation of the p53 tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers linked to a chemoresistance phenotype are unknown. Knowledge of the mechanism involved in the conversion of p53 is also sparse. Here, we report the massive accumulation of a p53 mutant within amyloid-like p53 oligomers in brain cells from glioblastoma tumors leading into chemoresistance gain of function. This finding, along with statistical analysis of data 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 suggest the role of amyloid-like mutant p53 oligomers as participants in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on new therapeutic options to avert 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 redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation ( E 1 / 2 ) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P 50 from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.