STI1 UNDERGOES LIQUID-LIQUID PHASE SEPARATION AND PREVENTS PRION PROTEIN AGGREGATION

Liquid-liquid phase separation (LLPS) is a fundamental biological mechanism that contributes to cellular organization and the formation of membraneless compartments, facilitating the concentration and interaction of specific biomolecules. The prion protein (PrP) is known for its ability to undergo LLPS and phase transitions, which begin with LLPS, but under certain conditions, these liquid droplets evolve into a gel phase and subsequently into solid protein aggregates. The protein STI1 (Stress-Inducible Phosphoprotein 1) is a co-chaperone that binds to PrP on the plasma membrane, playing a crucial role in neuroprotection and neuritogenesis. The aim of this study is to evaluate whether STI1 undergoes LLPS homotypically and to investigate the changes in LLPS characteristics when PrP is added to the solution containing STI1. For this, we used two constructs of recombinant murine PrP (rPrP^23-231 and rPrP^90-231) and recombinant murine STI1 (rSTI1). The interaction of rPrP^23-231 with rSTI1 was confirmed by isothermal titration calorimetry, which showed a high binding affinity (Kd = 1.5 μM). In microscopy assays, we demonstrated that rSTI1 undergoes phase separation in vitro and that interaction with both rPrP constructs resulted in a significant increase in the number and size of droplets. Turbidity assays corroborated these findings, showing an increase in solution turbidity consistent with LLPS formation. Our thioflavin T aggregation kinetics data revealed that the presence of rSTI1 prevented the formation of rPrP^23-231 aggregates, suggesting a protective role of rSTI1 in rPrP^23-231 aggregation. Transmission electron microscopy analyses confirmed the difference in size and morphology of rPrP^23-231 aggregates in the presence of rSTI1. Additionally, bioinformatics predictions using the FuzPred tool corroborated the experimental data, indicating a propensity of STI1 for phase separation. These results showed for the first time that the rSTI1 undergoes phase separation and that interaction with rPrP enhances this effect. At the same time, the interaction of rPrP^23-231 with rSTI1 prevented the formation of rPrP^23-231 aggregates, shedding light on the possibility of LLPS as a potential protective mechanism.