Heparin Modulates Prion Protein Liquid-Liquid Phase Separation, protecting from induced aggregation

Transmissible spongiform encephalopathies (TSEs) are rare diseases associated with the prion protein (PrP), caused by the conversion of cellular PrP (PrPC) into its misfolded, aggregated form (PrPSc). Although the exact mechanism remains unclear, recent studies highlight the role of phase separation (PS) as a precursor to protein aggregation. Modulating PS may be key to controlling this process. Previous findings showed that heparin (Hep) induces transient PrPC aggregation in a pH-dependent manner, forming a complex resistant to both RNA- and PrPSc-induced conversion. This study investigates whether Hep-induced effects on PrP involve PS. Using recombinant full-length and truncated PrP, PS was evaluated with differential interference contrast, fluorescence microscopy, and FRAP. Hek293 cells expressing YFP-PrP at the membrane were used to examine Hep effects in cellulo. Recombinant PrP (rPrP23-231, rPrP90-231, rPrP121-231) formed droplets in the presence of Hep, more prominently at pH 5.5 than at 7.4. Droplet formation by rPrP121-231 occurred only at pH 5.5. These droplets displayed liquid-like behaviors, such as fusion, dripping, and wetting, and were sensitive to increasing NaCl concentrations. Heparin increased PrP mobility both at the cell membrane and in intracellular compartments. PS induced by Hep was reversible, forming soluble complexes. Remarkably, at 70 °C, Hep prevented PrP aggregation by favoring droplet formation. These PrP:Hep droplets were also resistant to RNA-induced aggregation. Hep thus induces PS without crowding agents and stabilizes PrP structure by dynamic interactions with both the N-terminal and, at low pH, the C-terminal domains. In cells, Hep altered PrP distribution and dynamics, suggesting that PS may occur under physiological conditions and affect PrP trafficking and aggregation. These findings underscore the role of extracellular cofactors like Hep in modulating PrP behavior in vivo. Characterizing these early events in PrP misfolding provides valuable insight for therapeutic strategies aimed at maintaining PrP in its native conformation.