title: The (1-63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
abstract: The transcriptional regulator p53 plays an essential role in tumor suppression. Accordingly, it is found mutated, and its activity is reduced, in many human cancers. Recent reports show that some cancers are characterized by a loss of function of wild-type p53, which, in several cases, accumulates in intracellular aggregates. Although the nature of such aggregates is still unclear, recent evidence indicates that the p53 C-terminal and core domains can undergo amyloid aggregation in vitro under mild denaturing conditions, although no information is available on the largely unstructured N-terminal transactivation domain. We therefore decided to investigate the amyloid propensity of the acidic unfolded 1-63 fragment of the transactivation domain, cloned, expressed, and purified from a bacterial strain. Here we show that, when exposed to acidic pH, the 1-63 fragment forms thioflavine T-positive aggregates whose amyloid nature was confirmed by Fourier transform infrared spectroscopy analysis, atomic force microscopy, and x-ray diffraction. These aggregates were shown to be cytotoxic to human SH-SY5Y cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, and caspase-3 activity assays. These results add new significant details to the picture describing the propensity of single domains of p53 to aggregate, further suggesting that, under suitable destabilizing conditions, the whole protein may aggregate into amyloid assemblies in vivo.
authors.affiliation: Department of Biochemical Sciences, University of Florence, 50134 Florence, Italy. stefania.rigacci@unifi.it <stefania.rigacci@unifi.it>
authors.forename: Stefania
authors.initials: S
authors.lastname: Rigacci
authors.forename: Monica
authors.initials: M
authors.lastname: Bucciantini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pesce
authors.forename: Alessandra
authors.initials: A
authors.lastname: Gliozzi
authors.forename: Andrea
authors.initials: A
authors.lastname: Berti
authors.forename: Massimo
authors.initials: M
authors.lastname: Stefani
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D052160:Benzothiazoles; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003603:Cytotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D014961:X-Ray Diffraction
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D003603:Cytotoxins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; C009462:thioflavin T