Liquid-liquid phase separation of full-length prion protein initiates conformational conversionin vitro

Abstract

What initiates the sporadic misfolding process of PrP C remains to be elucidated. Here, we show that rPrP undergoes LPS at the interface of ATPS of polyethylene glycol (PEG)/dextran; also, subsequent liquid-solid phase transition attributed to the maturation of β-sheet structure initiated the spontaneous conversion of rPrP into PrP Sc -like amyloid. The intrinsically disordered region of PrP, residues 23-89, and kosmotropic anions were essential for the overall reaction. As the conversion of the protein proceeds without providing kinetic energy in this in vitro model, the findings suggest that LPS is associated with the occurrence of prion diseases and provide a new understanding of prion emergence. AbstractIn prion diseases, the causative agent is thought to be solely composed of misfolded prion proteins (PrP Sc ). It has been shown that PrP Sc can convert host-encoded normal isoform of prion protein (PrP C ), but the spontaneous emergence of PrP Sc is still enigmatic. Certain amyloidogenic proteins have recently been shown to undergo liquid phase separation (LPS) and liquid-solid phase transition. These phenomena are now believed to play an important role in the initial step of pathological aggregation in neurodegenerative diseases. We investigated whether purified fulllength human recombinant prion protein (rPrP, residues: 23-231) refolded normally can directly convert into PrP Sc -like amyloid via LPS and subsequent phase transition. The proteins underwent LPS in an aqueous two-phase system (ATPS) composed of 9% polyethylene glycol (PEG; M.W. 6000) and 9% dextran (M.W. 180,000) with kosmotropic anions. The maturation of droplets could be monitored by microscopy because the droplets of rPrP were positively stained by thioflavin T (ThT) at the beginning of LPS. Notably, phase transition occurred within an hour, accompanied by the β-sheet transition of rPrP. The aged gels of rPrP acquired resistance to proteinase-K (PK) digestion and formed amyloids, as confirmed by Congo red staining. These overall reactions required the N-terminal region of PrP, amino acid residues 23-89, which is known as an intrinsically disordered region. Our results suggest that the aberrant phase transition may be associated with the sporadic emergence of prions, and our assay should be useful for understanding the in vitro generation of prions.

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

“…1 A ). Both before and after TEV cleavage, GFP fluorescence was evenly distributed, indicating that neither C1 nor C2 formed biomolecular condensates under these conditions, corroborating recent results ( 41 ). Of note, the lack of LLPS was not due to inefficient TEV cleavage ( Fig.…”

“…The ability of full-length PrP to form biomolecular condensates has been described recently ( 41 , 42 , 43 , 44 , 45 ); however, the underlying molecular mechanisms that drive the formation of liquid-like droplets remain unknown. In the present study, we analyzed phase separation of full-length PrP and its major proteolytic fragments N1, N2, C1, and C2.…”

Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition

“…Proteins that can undergo LLPS, such as FUS, TDP-43, and Tau protein, usually contain repetitive domains or intrinsically disordered regions (IDR) (54)(55)(56)(57). Numerous studies identified that a variety of amyloid aggregation-prone proteins initiate aggregations through LLPS (58)(59)(60)(61). α-Syn has three IDRs and multiple low-complexity domains.…”

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high–molecular weight complexes

“…In terms of equating in vitro/in silico results with in vivo results, these structural biology effects and their allelic hierarchy tally with endpoints scored in PrP-expressing RK13 cells, specifically the heightened formation of PrP immunoreactive puncta and detergent insoluble complexes in S3 cells grown in Cbl-containing cell medium. A recent and perhaps parallel avenue of work on PrP concerns liquid-liquid phase-separation (LLPS) effects, often studied with recombinant protein presented on glass surfaces (99)(100)(101). Notably, the PrP N-terminal regionthe site of the S3 allele amino acid substitutionshas been deduced as being important for phase separation effects (100,101), a result in accordance with possession of two polybasic regions (101), low sequence complexity and regular positioning of aromatic residues as per the "sticker and spacer" model of phase transitions (102,103).…”

“…A recent and perhaps parallel avenue of work on PrP concerns liquid-liquid phase-separation (LLPS) effects, often studied with recombinant protein presented on glass surfaces (99)(100)(101). Notably, the PrP N-terminal regionthe site of the S3 allele amino acid substitutionshas been deduced as being important for phase separation effects (100,101), a result in accordance with possession of two polybasic regions (101), low sequence complexity and regular positioning of aromatic residues as per the "sticker and spacer" model of phase transitions (102,103). It is also striking that the PrP paralog protein shadoo has an N-terminal low complexity region, in this case an RGG repeat domain (104,105), with this class of proteins being well-accepted as undergoing LLPS (106).…”