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

Abstract

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 nativelike 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.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 PrP C into the misfolded form PrP Sc (1). Prion proteins are also present in yeast cells. Saccharomyces cerevisiae contains at least four prion-like proteins, including Ure2p, which is found aggregated intracellularly under certain conditions. However, 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). Such a change of phenotype could provide an evolutionary advantage to yeast cells by regulating nitrogen catabolism (5). When sources rich in nitrogen, such as ammonia, are available, Ure2p is believed to down-regulate the expression of gene products involved in the use of nitrogen-poor sources by binding the transcription activator Gln3p and preventing its migration into the nucleus (6). 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). This variant of Ure2...

Direct characterization of amyloidogenic oligomers by single-molecule fluorescence

“…This value falls within the broad range of aggregate sizes observed by TEM in the early stages of the reaction [see supporting information (SI) Text and Figs. S1-S4] for this and other systems (9,(22)(23)(24)(25). Taken together, the marked differences in the kinetic profiles of the aggregation reaction acquired from the ThT and sedimentation data strongly suggest the existence of multiple processes on the reaction pathway.…”

Amyloid Features and Neuronal Toxicity of Mature Prion Fibrils Are Highly Sensitive to High Pressure

“…Indeed, although several reports suggest that fibrils themselves possess toxicity (54,(77)(78)(79), oligomeric species are considered to be the primary cytotoxic species. Here we show that pressure treatment of amyloid fibrils results in partial structural changes and dissociation concomitantly with a strongly reduced cytotoxicity when compared with initial PrP fibrils.…”

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

“…This structure interacts with cell membrane, forming a water-filled channel of about 15 nm in diameter, large enough to permit the diffusion of small ions. On the other hand, native-like assemblies of yeast prion Ure2p retaining the a-helix content of the soluble monomer have been recently reported to be highly toxic to cultured cells (Pieri et al 2006).…”