Conversion of wild-type p53 core domain into a conformation that mimics a hot-spot mutant.

The wild-type p53 protein can be driven into a conformation corresponding to that adopted by structural mutant forms by heterodimerization with a mutant subunit. To seek partially folded states of the wild-type p53 core domain (p53C) we used high hydrostatic pressure (HP) and subzero temperatures. Aggregation of the protein was observed in parallel with its pressure denaturation at 25 and 37 degrees C. However, when HP experiments were performed at 4 degrees C, the extent of denaturation and aggregation was significantly less pronounced. On the other hand, subzero temperatures under pressure led to cold denaturation and yielded a non-aggregated, alternative conformation of p53C. Nuclear magnetic resonance (1H15N-NMR) data showed that the alternative p53C conformation resembled that of the hot-spot oncogenic mutant R248Q. This alternative state was as susceptible to denaturation and aggregation as the mutant R248Q when subjected to HP at 25 degrees C. Together these data demonstrate that wild-type p53C adopts an alternative conformation with a mutant-like stability, consistent with the dominant-negative effect caused by many mutants. This alternative conformation is likely related to inactive forms that appear in vivo, usually driven by interaction with mutant proteins. Therefore, it can be a valuable target in the search for ways to interfere with protein misfolding and hence to prevent tumor development.

Fibrillar aggregates of the tumor suppressor p53 core domain.

Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.