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J. Biol. Chem., Vol. 281, Issue 22, 15337-15344, June 2, 2006

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The Yeast Prion Ure2p Native-like Assemblies Are Toxic to Mammalian Cells Regardless of Their Aggregation State^*

Laura Pieri, Monica Bucciantini, Daniele Nosi, Lucia Formigli, Jimmy Savistchenko^¶, Ronald Melki^¶1, and Massimo Stefani^||2

From the Department of Biochemical Sciences, ^||Interuniversity Centre for the Study of the Molecular Basis of Neurodegenerative Diseases, and Department of Anatomy, Histology, and Forensic Medicine, University of Florence, Florence 50134, Italy and ^¶Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-Sur-Yvette Cedex, France

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the -helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.

Received for publication, October 27, 2005 , and in revised form, March 9, 2006.

^* This work was supported by Italian Ministero dell'Istruzione, dell'Università e della Ricerca Grants 2003054414 and RBNE01S29H_004 and the French Ministry of Education and Research through the Groupement d'Interêt Scientifique Prions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence may be addressed. E-mail: melki{at}lebs.cnrs-gif.fr. ² To whom correspondence may be addressed: Dept. of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy. Tel.: 39-055-4598307; Fax: 39-055-4598905; E-mail: stefani{at}scibio.unifi.it.

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