An Intrinsically Disordered Pathological Variant of the Prion Protein Y145Stop Transforms into Self-Templating Amyloids via Liquid-Liquid Phase Separation

Abstract

AbstractBiomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is thought to govern critical cellular functions, whereas, aberrant phase transitions are associated with a range of deadly neurodegenerative diseases. Here we show, a naturally occurring pathological truncation variant of the prion protein (PrP) by a mutation of a tyrosine residue at 145 to a stop codon (Y145Stop) yielding a highly disordered N-terminal IDR that spontaneously phase-separates into liquid-like droplets. Phase separation of this N-terminal segment that is rich in positively charged and aromatic residues is promoted by the electrostatic screening and a multitude of other transient, intermolecular, noncovalent interactions. Single-droplet Raman measurements in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies revealed that the intrinsic disorder and dynamics are retained in the liquid-like condensates. Lower concentrations of RNA promote the phase transition of Y145Stop at low micromolar protein concentrations under physiological condition. Whereas, higher RNA to protein ratios inhibit condensation indicating the role of RNA in modulating the phase behavior of Y145Stop. Highly dynamic liquid-like droplets eventually transform into dynamically-arrested, ordered, β-rich, amyloid-like aggregates via liquid-to-solid transition upon aging. These amyloid-like aggregates formed via phase separation display the self-templating characteristic and are capable of recruiting and autocatalytically converting monomeric Y145Stop into amyloid fibrils. In contrast to this disease-associated intrinsically disordered Y145 truncated variant, the wild-type full-length PrP exhibited a much lower propensity for phase separation and liquid-to-solid maturation into amyloid-like aggregates hinting at a potentially crucial, chaperone-like, protecting role of the globular C-terminal domain that remains largely conserved in vertebrate evolution. Such an intriguing interplay in the modulation of the protein phase behavior will have much broader implications in cell physiology and disease.