RT Journal Article SR Electronic T1 Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.01.25.919340 DO 10.1101/2020.01.25.919340 A1 Hiroya Tange A1 Daisuke Ishibashi A1 Takehiro Nakagaki A1 Yuzuru Taguchi A1 Yuji O. Kamatari A1 Hiroki Ozawa A1 Noriyuki Nishida YR 2020 UL http://biorxiv.org/content/early/2020/07/14/2020.01.25.919340.abstract AB Prion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in the spontaneous conversion remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation in the absence of PrPSc. We found that rPrP underwent liquid-liquid phase separation at the interface of the aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired proteinase-resistant amyloid accompanied by β-sheet conversion, as confirmed by western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote liquid-liquid phase separation. Thus, structural conversion via liquid–liquid phase separation and liquid–solid phase transition are intermediate steps in the conversion of prions.Competing Interest StatementThe authors have declared no competing interest.