THE (1-63) REGION OF THE p53 TRANSACTIVATION DOMAIN AGGREGATES IN VITRO INTO CYTOTOXIC AMYLOID ASSEMBLIES
Stefania Rigacci 1, Monica Bucciantini 1*, Annalisa Relini 2, Alessandra Pesce 3, Alessandra Gliozzi 3, Andrea Berti 4 and Massimo Stefani 4
1 university of Florence
2 university of Genoa
3 University of Genoa
4 University of Florence
* To whom correspondence should be addressed. E-mail: monica.bucciantini{at}unifi.it.
Submitted on September 19, 2007
Revised on October 15, 2007
Accepted on 26 November 2007
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Abstract |
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The transcriptional regulator p53 plays an essential role in tumour suppression. Accordingly, it is found mutated and its activity is reduced in many human cancers. Recent reports show that some cancers are characterised by a loss of function of wild type p53 which, in several cases, accumulates in intracellular aggregates. Though 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 while no information is available on the largely unstructured N-terminal transactivation domain. We then 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 FT-IR analysis, atomic force microscopy and X-ray diffraction. These aggregates were shown to be cytotoxic to human SH-SY5Y cells by MTT 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.
Key Words:
aggregation protein, amyloid, cancer, cytotoxicity, p53-conformation, p53-mutation
