Lee Amanda S, Galea Charles, DiGiammarino Enrico L, Jun Bokkyoo, Murti Gopal, Ribeiro Raul C, Zambetti Gerard, Schultz Christian P, Kriwacki Richard W
Department of Structural Biology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
J Mol Biol. 2003 Mar 28;327(3):699-709. doi: 10.1016/s0022-2836(03)00175-x.
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.
野生型p53（p53tet-wt）的四聚化结构域以及与儿童肾上腺皮质癌（ACC）相关的p53突变体R337H（p53tet-R337H），在某些条件下可从可溶性天然状态转变为淀粉样纤维。圆二色性、傅里叶变换红外光谱以及刚果红和硫黄素T染色表明，当在pH 4.0和升高的温度下孵育时，p53tet-wt和p53tet-R337H会采用一种交替的β-折叠构象（分别为p53tet-wt-beta和p53tet-R337H-beta），这是淀粉样纤维的特征。电子显微镜照片显示，p53tet-wt（p53tet-wt-beta）和p53tet-R337H（p53tet-R337H-beta）的交替构象是超分子结构，最好描述为“分子带”。傅里叶变换红外光谱分析表明，淀粉样纤维形成的机制涉及p53tet-wtβ-链的展开，随后是α-螺旋的展开，最后是形成含β-链的结构，其他方法显示这些结构是淀粉样带。突变体p53tet-R337H形成淀粉样纤维的倾向明显更高。当在室温（22摄氏度）下孵育一个月时，p53tet-wt（pH 4.0）和p
