HOP/STIP1 MODULATES PRION PROTEIN PHASE SEPARATION THROUGH NTERMINAL-DEPENDENT CO-CONDENSATION

The prion protein (PrP) is widely known for its involvement in transmissible spongiform encephalopathies when misfolded and aggregated in the scrapie form. However, its biological function in the native cellular form (PrP^C) is still not fully understood. Evidence suggests that PrP acts as a dynamic platform at the cell surface, organizing multicomponent protein complexes able to modulate distinct signaling pathways. Our group and others have previously demonstrated in vitro the phase separation (PS) of recombinant PrP (rPrP), a physical process driven by multivalent interactions and associated, in cellular systems, with signal transduction. At the plasma membrane, PrP^C interacts with the cochaperone HOP/STIP1 (Stress-Inducible Phosphoprotein 1), triggering neuroprotective signaling pathways and promoting neuronal survival. In this context, we aimed to characterize, using biophysical and microscopy approaches, how recombinant STIP1 (rSTIP1) modulates the phase separation of full-length recombinant PrP (rPrP^23-231). Our results show that recombinant rSTIP1 displays an intrinsic propensity for PS, forming condensates under different buffer conditions, modulated by molecular crowding, ionic strength, and weak hydrophobic interactions. We further observed that rSTIP1 co-condenses with rPrP^23-231, promoting the formation of larger condensates in a process dependent on the N-terminal region of PrP and predominantly driven by electrostatic interactions. In addition, in HEK293T cells expressing PrP^C-YFP-GPI, rSTIP1 promoted PrP^C redistribution at the plasma membrane, favoring the formation of punctate membrane structures. Together, these findings indicate that rSTIP1 modulates the phase separation of rPrP^23-231 and the spatial organization of PrP^C at the plasma membrane, supporting a model in which STIP1 promotes local PrP clustering at the cell surface.