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

Abstract

Biomolecular 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.

Spatiotemporal Modulations in Heterotypic Condensates of Prion and α-Synuclein Control Phase Transitions and Amyloid Conversion

“…We next used a naturally occurring PrP deletion mutant PrP 23-144 (Y145Stop) which retains the disordered N-terminal tail of PrP and is devoid of the C-terminal folded domain. 62,63 Y145Stop exhibited a similar phase separation propensity as compared to full-length PrP in the presence of α-Syn, indicating the necessary and sufficient role of the N-terminal IDR of PrP in two-component LLPS with α-Syn (Fig. 3b, Fig.…”

“…PrP 23-144 (Y145Stop), PrP 112-231, and single cysteine variants of full-length PrP (W31C and W99C) were used as described previously. 63,90 Recombinant full-length human α-Syn (1-140) plasmid cloned in vector pT7.7 was transformed in BL21(DE3)pLysS. 91 α-Syn (1-102) (N103Stop) and α-Syn (1-132) (Y133Stop) were created using the full-length α-Syn plasmid.…”

“…91 Recombinant prion protein constructs were overexpressed and purified using previously published protocols. 63,90 The purified proteins were refolded using the PD10 column in 14 mM MES buffer, pH 6.8. Recombinant α-Syn constructs except 103Stop were purified using the anion-exchange chromatography as described previously.…”

Single-Droplet Surface-Enhanced Raman Scattering Decodes the Molecular Language of Liquid-Liquid Phase Separation

“…Therefore, this ratio is an indicator of solvent-mediated hydrogen bonding propensity of the phenolic (-OH) group and is a measure of the water accessibility of tyrosine residues 43 . The I 850 /I 830 ratio is typically ≥ 2 for a well-solvated tyrosine and this ratio was found to be ∼ 0.5 for FUS droplets indicating considerable solvent protection possibly due to the participation of tyrosine residues in π-π stacking and/or cation-π interactions in the dense phase 44 . Another important sidechain band is the tryptophan Raman band typically observed at 880 cm -1 that arises due to the indole N-H bending and is often used to probe the environment and is a measure of the hydrogen bonding strength between the N-H of the indole ring with the surrounding solvent molecules.…”