ZINC IONS DRIVE THE PRION PROTEIN BIOMOLECULAR CONDENSATION AND MODULATE AGGREGATION

Prion diseases are characterized by the misfolding and conversion of the monomeric prion protein (PrP) to a multimeric aggregated pathogenic form, known as PrP^Sc. We have recently shown that biomolecular condensates formed via phase separation of PrP can undergo maturation to solid-like species resembling pathological aggregates, as in the presence of oxidative stress conditions. Conversely, the most well-studied ligand of PrP, copper ions, induce in vitro condensates of PrP that remain liquid-like over time, and accumulate Cu²⁺. Furthermore, live cells expressing PrP^C-EYFP show condensation at cell-cell interfaces as triggered by Cu²⁺. PrP can also bind Zn²⁺ ions via its intrinsically disordered octarepeats N-term domain, albeit with different affinities and binding modes as compared to Cu²⁺. Therefore, we hypothesized that Zn²⁺ could modulate PrP phase behavior differently from copper ions. Using a physiological-like buffer, we show that recombinant PrP undergoes phase separation with Zn²⁺. Furthermore, we show that metal ion-induced condensation of PrP is mediated by the N-term domain (residues 23-90). Cu²⁺ and Zn²⁺ promote liquid-like PrP condensates as shown by Fluorescence Recovery After Photobleaching. However, PrP:Zn²⁺ condensates exhibit a faster recovery. Cu²⁺ markedly inhibited seed-induced PrP aggregation kinetics, whereas Zn²⁺ provides a milder delay in PrP aggregation as probed by ThT assays. Our findings provide insights on Zn²⁺- and Cu²⁺-induced phase separation of PrP, supporting previously proposed functions of PrP in metal sequestering and uptake, processes that could be effectively regulated through cooperative assembly in biomolecular condensates.