FERRIC IONS MODULATE THE PRION PROTEIN PHASE SEPARATION

The aggregated form of the prion protein (PrP) is associated with the development of prion diseases. Evidence suggests that the cellular PrP participates in iron homeostasis by functioning as a ferrireductase, reducing Fe³⁺ to Fe²⁺ via its N-terminal octarepeats domain, that in turn facilitates cellular iron uptake. Furthermore, iron homeostasis is known to be dysregulated in the brains of transgenic mice overexpressing PrP. Recently, our group reported that metal ions such as Cu²⁺ and Zn²⁺ induce the formation of PrP biomolecular condensates, whereas oxidizing conditions favor its aggregation. Our central goal is to investigating the impact of Fe³⁺ ions on PrP phase transitions using an in vitro reconstituted system with recombinant murine PrP. The recombinant full-length PrP was expressed in E. coli and purified by nickel-affinity chromatography. Ferric ions were able to trigger the formation of biomolecular condensates in 25 µM PrP solutions; these condensates exhibited liquid-like material properties at a 1:1 molar ratio (protein:Fe³⁺), as observed by phase-contrast microscopy. In aggregation assays, after 72 hours of incubation, epifluorescence microscopy using Thioflavin T (ThT) revealed aggregates that tested positive for this fluorescent probe, indicating the formation of β-sheet-rich structures in samples incubated with Fe3+. Aggregate formation was observed at a 1:1 stoichiometry (protein:Fe³⁺) compared to the control. Quantitative extrinsic fluorescence assays using ThT confirmed a significant increase in fluorescence intensity for the condition containing 8× molar excess of Fe³⁺ in the absence of PEG4K; however, samples containing PEG4K did not exhibit this increase. Further experiments will be conducted, including ThT-based aggregation kinetics assays, transmission electron microscopy, and fluorescence recovery after photobleaching (FRAP). Our preliminary data suggest that Fe³⁺ triggers the formation of PrP condensates under near-physiological conditions, with the properties of these condensates are modulated by Fe³⁺ concentration. This study provides preliminary insights into iron-driven PrP condensation and aggregation, and suggests that phase separation might represent a modulable mechanism with potential to inhibit or delay PrP aggregation.