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.
Article: Early and late cytotoxic effects of external application of the Alzheimer's Abeta result from the initial formation and function of Abeta ion channels.[show abstract] [hide abstract]
ABSTRACT: Extracellular application of the Alzheimer's beta-amyloid (Abeta) peptide evokes a series of cellular responses that leads to the death of cells by apoptosis. Some responses to freshly prepared Abeta occur immediately, including changes in intracellular calcium concentration and changes in membrane permeability and phosphatidylserine asymmetry. We show here that the cytotoxic action of externally applied Abeta, such as caspase activation and apoptotic loss of cell viability, occurs and persists even several days after Abeta is removed from the medium. We find that the mechanism for this persistent cytotoxic action of extracellular Abeta is based on the sustained activity of active Abeta ion channels that remain incorporated in the cell membrane. To confirm this assessment, we blocked the late cytotoxic action of Abeta with the classically known Abeta channel blockers zinc and tromethamine. To further validate this conclusion, we developed a specific peptide segment from the sequence forming the mouth of the Abeta channel to block Abeta Ca2+ channels acutely and to block late Abeta effects on caspase activation and apoptosis. This is the first report of a specific Abeta channel blocker compound, NA4, which efficaciously and potently blocks the most known cellular responses to Abeta.Biochemistry 06/2006; 45(18):5907-15. · 3.42 Impact Factor