HEPARIN MODULATES PRION PROTEIN LIQUID-LIQUID PHASE SEPARATION PROTECTING FROM TEMPERATURE-INDUCED AGGREGATION

Transmissible spongiform encephalopathies (TSEs) are rare diseases linked to the prion protein (PrP). These diseases occur due to the conversion of cellular PrP (PrP^C) into scrapie PrP (PrPSc) and subsequent aggregation, though the precise mechanism is still unclear. Recent research suggests that preliminary phase separation (PS) events contribute to protein aggregation, making the modulation of these events critical in controlling aggregation. Previous studies have shown that heparin (Hep) induces transient aggregation of PrP^C in a pH-dependent manner, and the PrP:Hep complex resists RNA-induced and PrP^Sc-seeded conversion. This study explores whether the observed effect of Hep is associated with the PS process and examines the impact of temperature and pH on Hep-induced PS. Using recombinant full-length mouse PrP^C23-231 and truncated rPrP90-231, the study evaluates the importance of the PrP N-terminal domain. Different PrP concentrations in the presence of heparin were assessed using Differential Interference Contrast (DIC), Fluorescence Recovery After Photobleaching (FRAP), and fluorescence microscopy. At room temperature, both rPrP23-231 and rPrP90-231 phase-separated in the presence of Hep, with higher PrP concentrations leading to larger droplets. The number of droplets was more significant at pH 5.5 compared to 7.4, and rPrP90-231 droplets formed robustly only at higher protein concentrations and pH 5.5. The experiments confirmed the liquid-liquid nature of the droplets through observations of droplet fusion, dripping, and surface-welting events. Increasing NaCl concentrations prevented droplet formation, and fluorescence confocal microscopy verified the colocalization of PrP and heparin within the droplets. The phase separation induced by heparin was reversible, resulting in a soluble PrP:Hep complex At 70 °C, the high temperature induced rapid PrP aggregation. However, it led to the formation of PrP:Hep liquid droplets. Heparin alone triggered PS of PrP without molecular crowding agents, highly dependent on Hep's interaction with the protein's flexible N-terminal domain. Interaction with the C-terminal globular domain at acidic pH also induced PS. The dynamic nature of PrP:Hep and PrP:PrP contacts protected PrP from temperature-induced aggregation, forming droplets and sustaining PrP structural stability. Understanding these precursor states and modulators of PrP misfolding pathways provides essential information for evaluating treatments aimed at stabilizing the native form of PrP.