STI1 Phase Separates and Modulates PrP Droplet Behavior

The prion protein (PrP) plays a central role in neurodegenerative diseases due to its ability to undergo conformational conversion and aggregation. PrP undergoes liquid-liquid phase separation (LLPS), forming dynamic droplets that can fulfill physiological roles but can also evolve into pathogenic aggregates. At the plasma membrane, PrP interacts with the co-chaperone HOP/STI1 (Stress-Inducible Phosphoprotein 1), which triggers neuroprotective signaling pathways and promotes neuronal survival. Here, we investigated how STI1 modulates the phase behavior of PrP. We used isothermal titration calorimetry to confirm the interaction between recombinant murine STI1 (rSTI1) and full-length PrP (rPrP23–231), with micromolar affinity. LLPS was assessed using predictive modeling (FuzPred), phase-contrast microscopy, turbidity measurements, X-ray photon correlation spectroscopy (XPCS), and fluorescence recovery after photobleaching (FRAP). rSTI1 undergoes LLPS homotypically in vitro, as predicted by FuzPred and demonstrated by microscopy and turbidity assays. Remarkably, when rSTI1 and rPrP are combined in conditions where neither protein phase-separates alone, LLPS is induced. Under LLPS-permissive conditions, their co-assembly results in significantly larger and more abundant droplets. Disruption by NaCl and partial sensitivity to 1,6-hexanediol indicate a combination of electrostatic and hydrophobic forces driving droplet formation. Droplet dynamics were assessed using XPCS and FRAP, both of which revealed alterations in droplet dynamics upon interaction between rSTI1 and rPrP. These findings suggest a novel role for STI1 in modulating PrP LLPS and may represent a protective mechanism to sequester PrP into dynamic, non-aggregating structures, preventing its pathological conversion.

Support: FAPERJ, CNPq, CAPES