Vitronectin peptides and NCAM induce prion protein phase separation and modulate its aggregation

The prion protein (PrP) is a glycosylphosphatidylinositol-anchored glycoprotein on the outer face of the plasma membrane, primarily expressed in the central nervous system. Although its biological function is not fully elucidated, it acts as a multicomponent interaction platform on the cell membrane, binding to various partners to activate signaling pathways. Among these ligands are vitronectin and the neural cell adhesion molecule (NCAM), whose mapped interaction sites correspond to residues 309–322 (vitronectin) and 620–635 (NCAM, BCL region), which are associated with the induction of neuritogenesis and axonal growth. PrP can undergo homotypic or heterotypic phase separation (PS), a process potentially facilitated by its multiple interactions on the cell surface. Our objective is to thermodynamically characterize the interaction of full-length recombinant PrP (rPrP^23-231) with synthetic vitronectin and NCAM peptides, investigating its structural consequences, the potential to induce PS, and the effects on protein aggregation. To this end, we employed isothermal titration calorimetry (ITC) to evaluate binding affinity; circular dichroism (CD) to analyze secondary structure; phase-contrast and fluorescence microscopy, combined with turbidimetry assays, to observe and quantify PS; and assays with the thioflavin T (ThT) probe to monitor aggregation kinetics. ITC results indicated that rPrP^23-231 interacts with high affinity with both peptides, displaying Kd values in the micromolar range. CD analyses showed that the secondary structure of rPrP^23-231 remains stable in the presence of the ligands. Microscopy images and turbidimetry convergently demonstrated that both peptides induce PS of rPrP^23-231. Regarding aggregation kinetics, vitronectin inhibited rPrP^23-231 aggregation, whereas the NCAM peptide reduced the lag phase of the process. Taken together, these results indicate that vitronectin and NCAM peptides promote the phase separation of rPrP^23-231 and modulate its aggregation without altering its secondary structural conformation. In the future, we intend to complement these data with FRAP (Fluorescence Recovery After Photobleaching), DLS (Dynamic Light Scattering) assays, and simultaneous interaction studies of rPrP^23-231 with both peptides.