Abstract
The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-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.
Highlights
It is widely accepted that transmissible spongiform encephalopathies, a group of neurodegenerative diseases in mammals, are due to misfolding of the infectious prion protein (PrP)3; when altered, the protein is able to promote the conformational conversion of the normal cellular form PrPC into the misfolded form PrPSc [1]
Unlike mammalian prions, which display cytotoxicity in their aggregated form, Ure2p appears substantially noncytotoxic when aggregated into yeast cells [2], providing the molecular explanation for the change of phenotype given by the nonchromosomal genetic element [URE3] [3, 4]
In wild-type cells, Ure2p is dispersed in the cytoplasm, whereas the [URE3] phenotype is caused by the appearance of an altered, self-propagating form of Ure2p (Ure2p[URE3]) able to form in the cytoplasm large globular or elongated aggregates [4]
Summary
It is widely accepted that transmissible spongiform encephalopathies, a group of neurodegenerative diseases in mammals, are due to misfolding of the infectious prion protein (PrP); when altered, the protein is able to promote the conformational conversion of the normal cellular form PrPC into the misfolded form PrPSc [1]. Unlike mammalian prions, which display cytotoxicity in their aggregated form, Ure2p appears substantially noncytotoxic when aggregated into yeast cells [2], providing the molecular explanation for the change of phenotype given by the nonchromosomal genetic element [URE3] [3, 4]. Ure2p assembles in vitro into oligomers, eventually giving rise to fibrils similar to those observed in yeast cells carrying the [URE3] phenotype [7, 9] These fibrils share several morphological, structural, and tinctorial features with amyloids, including enhanced resistance to proteolysis [7, 9, 16], increased thioflavin-T fluorescence, and the yellow-green birefringence in cross-polarized light upon Congo red binding [17]. These fibrils lack the classical x-ray diffraction pattern characteristic of amyloids, and their constituent monomers maintain the native structure rich in ␣-helices, can bind GSH [16, 18], and show gluta-
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