Prion protein through the lens of liquid phase separation

Proteins are the essential constituents of all living organisms which keep the cells under constant surveillance. The phenomenal diversity of cellular functions performed by these proteins is in turn dictated by their specific three-dimensional structure. However, acquiring this correctly folded conformation in a highly crowded cellular environment is highly error-prone and can often give rise to incorrectly folded proteins/misfolded proteins. Such protein misfolding is also a common feature in several aging and neurodegenerative disorders, for instance, Alzheimer’s, Parkinson’s, Huntington’s, prion diseases, and so forth. This good to evil transformation of protein from its functional native state to the misfolded disease-causing state can be imagined as the split personality of the character in the novel The Strange Case of Dr. Jekyll and Mr. Hyde.

The misfolding is often also assisted by multiple binding partners like nucleic acids, proteins, salts, and so forth. Also, there is increasing evidence of the presence of unrelated/multiple proteins in these amyloid-like aggregates which indicates cross-interaction between multiple proteins. Therefore, the central idea behind these projects was to decode the molecular mechanism of the protein misfolding as well as to identify the factors which promote such events. 

We elucidated a novel mechanism by which a pathological variant of the prion protein retaining only the N-terminal intrinsically disordered region (IDR) misfolds and forms amyloid-like aggregates. At higher concentrations, the protein undergoes spontaneous biomolecular condensation/liquid-liquid phase separation (LLPS) to form liquid-like protein-rich condensates. The addition of certain cofactors like nucleic acids promoted phase separation at physiological protein concentrations. We also characterized different features that regulate prion condensation. The N-terminal IDR and the sequence-specific bias of the prion protein promote its LLPS. These condensates are highly dynamic and reversible i.e., they respond to various external factors like temperature, pH, etc. However, upon aging, these condensates are no longer reversible and exhibit solid-like behavior. Our study showed that these protein-rich droplets might act as the center for the growth and maturation of amyloid-like fibrils which are a common occurrence in prion diseases. 

In another study, we also demonstrate the heterotypic phase separation of native prion protein (Fl-PrP) with another protein namely α-synuclein which occurs at much lower protein concentrations. Domain-specific electrostatic interactions between the N-terminal of Fl-PrP and C-terminal of α-synuclein facilitate their condensation whereas the addition of RNA in a certain ratio resulted in their dissolution. These heterotypic condensates upon aging also demonstrate similar liquid-to-solid transition indicating a central role of such phase transition pathways in the onset of neurodegenerative disorders.