Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337Hbeta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt P-strands, followed by unfolding of the a-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degreesC) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degreesC over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells. (C) 2003 Elsevier Science Ltd. All rights reserved.