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-R337H-beta) 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 beta-strands, followed by unfolding of the alpha-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 degrees C) 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 degrees C 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.
Lee
Amanda S
AS
Department of Structural Biology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
Galea
Charles
C
DiGiammarino
Enrico L
EL
Jun
Bokkyoo
B
Murti
Gopal
G
Ribeiro
Raul C
RC
Zambetti
Gerard
G
Schultz
Christian P
CP
Kriwacki
Richard W
RW
D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D002942:Circular Dichroism; D004396:Coloring Agents / Q000494:pharmacology; D003224:Congo Red / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics*
D000682:Amyloid; D052160:Benzothiazoles; D004396:Coloring Agents; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; C009462:thioflavin T; D003224:Congo Red; D019791:Guanidine