title: Synthetic lipid vesicles recruit native-like aggregates and affect the aggregation process of the prion Ure2p: insights on vesicle permeabilization and charge selectivity.
abstract: The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.
authors.affiliation: Department of Biochemical Sciences, University of Florence, Italy; Research Centre on the Molecular Basis of Neurodegeneration, University of Florence, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Pieri
authors.forename: Monica
authors.initials: M
authors.lastname: Bucciantini
authors.forename: Patrizio
authors.initials: P
authors.lastname: Guasti
authors.forename: Jimmy
authors.initials: J
authors.lastname: Savistchenko
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
authors.forename: Massimo
authors.initials: M
authors.lastname: Stefani
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002712:Chlorides / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005979:Glutathione Peroxidase; D008564:Membrane Potentials; D051379:Mice; D046529:Microscopy, Electron, Transmission; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D010784:Photomicrography; D011328:Prions / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D013844:Thiazoles / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; D002712:Chlorides; D005452:Fluoresceins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; D053835:Unilamellar Liposomes; C009462:thioflavin T; D002784:Cholesterol; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D002118:Calcium; C007740:fluorexon