PHOSPHATIDIC ACID VESICLES INDUCE N-TERMINAL-INDEPENDENT AMYLOID AGGREGATION OF PRION PROTEIN IN A TEMPERATURE-DEPENDENT MANNER

INTRODUCTION: Conversion of the cellular prion protein (PrP^C) into its pathogenic assemblies (prion scrapie, PrP^Sc) is the central molecular event underlying prion diseases. Biomolecular cofactors, including nucleic acids and membrane lipids, have been implicated in this process, although the mechanisms by which individual lipids promote PrP conversion remain poorly understood. Previous work from our group demonstrated that phosphatidic acid (PA) vesicles induce aggregation of murine recombinant PrP (recPrP) in the absence of additional cofactors. OBJECTIVES: Here, we aimed to characterize the molecular pathway of PA-induced PrP aggregation and investigate the roles of membrane physical state during this process. METHODS: Spectroscopic techniques were used to monitor amyloid formation and aggregate stability over a range of temperatures. Full-length recPrP and an N-terminally truncated construct were compared to evaluate the contribution of the N-terminal region to PA-induced conversion. Microscopy techniques were employed to assess aggregate morphology, and SDS-PAGE analysis was used to evaluate the proteinase K (PK) resistance of the aggregates. RESULTS AND DISCUSSION: PA vesicles promoted amyloid aggregation of both PrP constructs with comparable efficiency, indicating that most of the N-terminal domain is dispensable for PA-mediated conversion. Thermal stability analysis revealed the formation of highly thermostable β-sheet-rich fibrils, while suggesting that higher-order fibril packing remains sensitive to the presence of PA membranes. Fibrillization was favored at lower temperatures, where PA bilayers are more rigid, whereas PrP alone exhibited no detectable temperature-dependent structural changes. This indicates that membrane organization, rather than intrinsic protein stability, governs the efficiency of PA-induced conversion. Transmission electron microscopy (TEM) confirmed the formation of amyloid fibrils under these conditions. PrP fibrils displayed partial resistance to proteinase K (PK) digestion, and fragmentation of the fibrils revealed a cytotoxic effect. CONCLUSIONS: Our findings demonstrate that PA promotes N-terminal-independent PrP aggregation and that the physical state of the membrane is a critical determinant of fibrillization efficiency. These results establish PA vesicles as a valuable model for investigating the early molecular events of cofactor-driven prion conversion and provide new insights into how membrane properties regulate pathological protein aggregation.