PHASE SEPARATION RESEARCH APPLIED TO THE STUDY OF THE BIOLOGICAL FUNCTION OF THE PRION PROTEIN

The conversion of the prion protein (PrP) into its pathological scrapie form (PrPSc), followed by its subsequent accumulation is the attributed cause of certain incurable neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Although the biological function of the prion protein (PrP) is still unknown, putative functions are based on PrP-interacting molecules present on the cell surface, such as the stress-inducible phosphoprotein 1 (STI1), the laminin receptor precursor (LRP) and the neural cell adhesion molecule (NCAM). It is currently considered that PrP is a cell surface scaffolding protein for a variety of signaling modules, which can be allosterically regulated and triggers differing functional consequences. Recombinant murine PrP (rPrP) undergoes liquid-liquid phase separation (LLPS) in vitro and the multivalency of interactions involving PrP suggests that LLPS occurs in the plasma membrane, a process associated with signal transduction. Our goal is to evaluate the interaction of multiple protein ligands with PrP and try to correlate the interaction events with the biological function of PrP. For the in vitro assays, we used two rPrP constructs (rPrP23-231 and rPrP90-231), recombinant STI1 (His6-STI1) and synthetic peptides from STI1, NCAM and LRP, previously described as PrP ligands. For the in silico assays, we used a coarse grained model of full-length PrP and peptide fragments from STI1, LRP and NCAM. The simulations were performed in the GROMACS 2022.3 program using the AMBER99SB-disp force field. The interaction of rPrP with His6-STI1 and with the synthetic peptides was confirmed by isothermal titration calorimetry, where differences in affinity and in the thermodynamic parameters of the interaction between the two rPrP constructs were observed. We used molecular dynamics to observe the molecular effects of peptide interaction on PrP dynamics and we found that these ligands modulate PrP differently, suggesting allosteric effects on rPrP as a result of interaction with these ligands. Through confocal microscopy, we observed that rPrP phase-separates in the presence of peptides and co-localizes with them within droplets. Using phase contrast microscopy, it was seen that His6-STI1 phase separated and that association with rPrP resulted in LLPS in solution. We next evaluated through thioflavin T fluorescence assay and transmission electron microscopy the effect of this interaction on the rPrP aggregation process. We observed that the interaction of rPrP with His6-STI1 prevented the formation of rPrP aggregates. Finally, confocal microscopy assays showed the formation of PrP clusters in the plasma membrane of HEK 293T cells transfected with PrP-EYFP. Thus, our data provide experimental evidence that reinforces the proposal that PrP acts as a multicomponent interaction platform that can be allosterically modulated. Multivalent interactions with binding partners result in LLPS and further studies are needed to understand how this process is related to physiological and/or pathological processes.