A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP

Carola Munoz-Montesino,Djabir Larkem,Clément Barbereau,Angélique Igel-Egalon,Sandrine Truchet,Eric Jacquet,Naïma Nhiri,Mohammed Moudjou,Christina Sizun,Human Rezaei,Vincent Béringue,Michel Dron

doi:10.1074/jbc.ra120.014738

Carola Munoz-Montesino, Djabir Larkem + Show 10 more

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https://doi.org/10.1074/jbc.ra120.014738

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Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Highlights

Mammalian prions are responsible for transmissible spongiform encephalopathies in both humans and animals
We show that specific deletion of the larger H2 C-terminal segment HTVTTTT, which removes three additional residues, causes the spontaneous conversion of the mutant ovine prion protein (PrP) into a new type of prion
This is in agreement with NMR analysis of the segment 113– 214 of D190–196 PrP (C1113), which contains the entire sequence of the structured domain and is an equivalent to the natural C1 fragment studied hereinafter

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Mammalian prions are responsible for transmissible spongiform encephalopathies in both humans and animals. C1 is smaller than C2 and considered so far too short to be converted into prions, it encompasses the structured globular domain of the full-length PrPC and is present at the cell surface [13, 14]. Prions can emerge spontaneously as in sporadic cases of human Creutzfeldt–Jakob disease (CJD), that is, without evidence of infection or contamination. In this context, prion generation requires at first the formation of nuclei stable enough to initiate the polymerization process, which is expected to be a slow and rate-limiting step [15]. More than 30 mutations responsible for inherited human prion

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