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• W3048482863 abstract "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. 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. Mammalian prions are responsible for transmissible spongiform encephalopathies in both humans and animals. Prions result from the misfolding of the host-encoded prion protein (PrP). Under its normal conformation, the cell-surface GPI-anchored PrP (PrPC) presents a globular domain containing three α-helices and two short antiparallel β strands, preceded by an unstructured N-terminal part (1Prusiner S.B. Biology and genetics of prions causing neurodegeneration.Annu. Rev. Genet. 2013; 47 (24274755): 601-62310.1146/annurev-genet-110711-155524Crossref PubMed Scopus (336) Google Scholar, 2Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. López García F. Billeter M. Calzolai L. Wider G. Wüthrich K. NMR solution structure of the human prion protein.Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (10618385): 145-15010.1073/pnas.97.1.145Crossref PubMed Scopus (948) Google Scholar). In contrast, prions are made from assemblies of β-sheet–rich, insoluble, aggregative, and mostly partially protease-resistant PrP conformers called PrPSc in reference to their original identification in scrapie-infected sheep (3Prusiner S.B. Novel proteinaceous infectious particles cause scrapie.Science. 1982; 216 (6801762): 136-14410.1126/science.6801762Crossref PubMed Scopus (4101) Google Scholar). Prion replication appears to proceed by conversion of the normal protein through templated polymerization (4Come J.H. Fraser P.E. Lansbury Jr., P.T. A kinetic model for amyloid formation in the prion diseases: importance of seeding.Proc. Natl. Acad. Sci. U.S.A. 1993; 90 (8327467): 5959-596310.1073/pnas.90.13.5959Crossref PubMed Scopus (354) Google Scholar), which explains not only their propagation in tissues but also their intra- or interspecific infectivity. The high-resolution structure of PrPSc is not yet resolved, because of inherent difficulties in producing large amounts of purified insoluble assemblies that may nonetheless have some intrinsic heterogeneity with respect to size. Several amyloid models were proposed, and coexistence of different candidate structures has even been suggested (5Groveman B.R. Dolan M.A. Taubner L.M. Kraus A. Wickner R.B. Caughey B. Parallel in-register intermolecular β-sheet architectures for prion-seeded prion protein (PrP) amyloids.J. Biol. Chem. 2014; 289 (25028516): 24129-2414210.1074/jbc.M114.578344Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 6Spagnolli G. Rigoli M. Orioli S. Sevillano A.M. Faccioli P. Wille H. Biasini E. Requena J.R. Full atomistic model of prion structure and conversion.PLoS Pathog. 2019; 15 (31295325): e100786410.1371/journal.ppat.1007864Crossref PubMed Scopus (74) Google Scholar, 7Baskakov I.V. Caughey B. Requena J.R. Sevillano A.M. Surewicz W.K. Wille H. The prion 2018 round tables (I): the structure of PrP(Sc).Prion. 2019; 13 (30646817): 46-5210.1080/19336896.2019.1569450Crossref PubMed Scopus (23) Google Scholar). The two-third C-terminal part of PrP forming the protease-resistant core of PrPSc, the C2 fragment, constitutes the domain necessary and sufficient for prion replication (8Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. Truncated forms of the human prion protein in normal brain and in prion diseases.J. Biol. Chem. 1995; 270 (7642585): 19173-1918010.1074/jbc.270.32.19173Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 9Fischer M. Rülicke T. Raeber A. Sailer A. Moser M. Oesch B. Brandner S. Aguzzi A. Weissmann C. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.EMBO J. 1996; 15 (8635458): 1255-126410.1002/j.1460-2075.1996.tb00467.xCrossref PubMed Scopus (780) Google Scholar). Prion strains are identified by their specific biochemical and/or neuropathological features in the same infected host species (10Rossi M. Baiardi S. Parchi P. Understanding prion strains: evidence from studies of the disease forms affecting humans.Viruses. 2019; 11 (30934971): 30910.3390/v11040309Crossref PubMed Scopus (29) Google Scholar, 11Dickinson A.G. Meikle V.M. Fraser H. Identification of a gene which controls the incubation period of some strains of scrapie agent in mice.J. Comp. Pathol. 1968; 78 (4970191): 293-29910.1016/0021-9975(68)90005-4Crossref PubMed Scopus (348) Google Scholar). Strains result from structural differences in three-dimensional or quaternary structure of PrPSc. The N-terminal border of the C2 fragment is strain-dependent and can vary around amino acid positions 80–100. A C-terminal fragment called C1 results from the natural cleavage of PrPC by a cellular protease at the α cleavage site, which is between residues 110 and 111 of human PrP (8Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. Truncated forms of the human prion protein in normal brain and in prion diseases.J. Biol. Chem. 1995; 270 (7642585): 19173-1918010.1074/jbc.270.32.19173Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 12Mangé A. Béranger F. Peoc'h K. Onodera T. Frobert Y. Lehmann S. Alpha- and beta-cleavages of the amino-terminus of the cellular prion protein.Biol. Cell. 2004; 96 (15050367): 125-13210.1016/j.biolcel.2003.11.007Crossref PubMed Scopus (140) Google Scholar). C1 is thus smaller than C2 and considered so far too short to be converted into prions, although it encompasses the structured globular domain of the full-length PrPC and is also present at the cell surface (13Shmerling D. Hegyi I. Fischer M. Blättler T. Brandner S. Götz J. Rülicke T. Flechsig E. Cozzio A. von Mering C. Hangartner C. Aguzzi A. Weissmann C. Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions.Cell. 1998; 93 (9568713): 203-21410.1016/S0092-8674(00)81572-XAbstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 14Westergard L. Turnbaugh J.A. Harris D.A. A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation.J. Biol. Chem. 2011; 286 (22025612): 44234-4424210.1074/jbc.M111.286195Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (15Cobb N.J. Surewicz W.K. Prion diseases and their biochemical mechanisms.Biochemistry. 2009; 48 (19239250): 2574-258510.1021/bi900108vCrossref PubMed Scopus (156) Google Scholar). Indeed, spontaneous prion disease is a rare event, the prevalence of sporadic CJD being of ∼1.5 cases per million per year worldwide. Mutations in PrP can favor PrP spontaneous conversion into prions. Indeed, more than 30 mutations responsible for inherited human prion diseases, including genetic CJD, Gerstmann–Sträussler–Scheinker syndrome (GSS), or fatal familial insomnia, were identified, and these dominant allelic mutations usually show a high penetrance (16Mead S. Lloyd S. Collinge J. Genetic factors in mammalian prion diseases.Annu. Rev. Genet. 2019; 53 (31537104): 117-14710.1146/annurev-genet-120213-092352Crossref PubMed Scopus (36) Google Scholar). Disease-causing mutations might favor partial unfolding or transient denaturation of PrPC, which are required for refolding into PrPSc, and might also increase stability of initial PrPSc seeds. The cellular and molecular processes underpinning or preventing spontaneous prion generation remain poorly understood. Transgenic mouse models of spontaneous prion formation have proven difficult to obtain. This was achieved for human or mouse PrP bearing some mutations (17Jackson W.S. Borkowski A.W. Watson N.E. King O.D. Faas H. Jasanoff A. Lindquist S. Profoundly different prion diseases in knock-in mice carrying single PrP codon substitutions associated with human diseases.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23959875): 14759-1476410.1073/pnas.1312006110Crossref PubMed Scopus (33) Google Scholar, 18Friedman-Levi Y. Meiner Z. Canello T. Frid K. Kovacs G.G. Budka H. Avrahami D. Gabizon R. Fatal prion disease in a mouse model of genetic E200K Creutzfeldt–Jakob disease.PLoS Pathog. 2011; 7 (22072968): e100235010.1371/journal.ppat.1002350Crossref PubMed Scopus (61) Google Scholar). Hallmarks of the disease were not always reproduced in mice, and intriguingly in several instances, prions showed a rather low resistance to protease digestion (19Watts J.C. Prusiner S.B. Experimental models of inherited PrP prion diseases.Cold Spring Harb. Perspect. Med. 2017; 7 (28096244): a02715110.1101/cshperspect.a027151Crossref PubMed Scopus (20) Google Scholar). Prions spontaneously formed in mice overexpressing either anchorless mouse PrP or I109 allele of bank vole PrP (20Stöhr J. Watts J.C. Legname G. Oehler A. Lemus A. Nguyen H.O. Sussman J. Wille H. DeArmond S.J. Prusiner S.B. Giles K. Spontaneous generation of anchorless prions in transgenic mice.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (22160704): 21223-2122810.1073/pnas.1117827108Crossref PubMed Scopus (57) Google Scholar, 21Watts J.C. Giles K. Bourkas M.E. Patel S. Oehler A. Gavidia M. Bhardwaj S. Lee J. Prusiner S.B. Towards authentic transgenic mouse models of heritable PrP prion diseases.Acta Neuropathol. 2016; 132 (27350609): 593-61010.1007/s00401-016-1585-6Crossref PubMed Scopus (31) Google Scholar). Currently, no cellular model for spontaneous prion formation has been reported. Toward this goal we focused here on an intriguing highly conserved threonine-rich region of the α-helix H2 associated with several disease-causing mutations in human PrP (22Dima R.I. Thirumalai D. Probing the instabilities in the dynamics of helical fragments from mouse PrPC.Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (15494440): 15335-1534010.1073/pnas.0404235101Crossref PubMed Scopus (107) Google Scholar). In a recent work, we demonstrated that deletion of the cluster of four threonines in the α-helix H2 C terminus has no or marginal effect on ovine prions replication in RK13 cells expressing ovine PrP (23Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Chapuis J. Ciric D. Igel-Egalon A. Laude H. Béringue V. Rezaei H. Dron M. Generating bona fide mammalian prions with internal deletions.J. Virol. 2016; 90 (27226369): 6963-697510.1128/JVI.00555-16Crossref PubMed Scopus (9) Google Scholar, 24Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Igel-Egalon A. Barbereau C. Chapuis J. Ciric D. Laude H. Béringue V. Rezaei H. Dron M. A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity.Prion. 2017; 11 (28281924): 25-3010.1080/19336896.2016.1274851Crossref PubMed Scopus (3) Google Scholar). We now 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 PrP into a new type of prion. This prion exhibits a main protease-resistant core shorter than usual, of C1 size, which was able to infect naïve RK13 cells expressing the mutant C1 segment alone. The potential importance of H2 C terminus for maintenance of normal PrPC conformation in the cell, the specificities of the new mutant prion, and the surprising conversion of the homologous mutant C1 fragment into a prion entity are discussed. We focused here on Δ190–196 ovine PrP (VRQ allelic variant), a mutant PrP with a specific deletion of seven amino acids at the end of helix H2 (Fig. 1). We previously reported that a larger deletion of the H2 C terminus (Δ190–197) did not have a major impact on the structure of the protein, leaving intact the spatial organization of the three α-helices in the globular domain of PrP (23Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Chapuis J. Ciric D. Igel-Egalon A. Laude H. Béringue V. Rezaei H. Dron M. Generating bona fide mammalian prions with internal deletions.J. Virol. 2016; 90 (27226369): 6963-697510.1128/JVI.00555-16Crossref PubMed Scopus (9) Google Scholar). As with Δ190–197 PrP, structural analysis of recombinant Δ190–196 PrP by CD indicated a conservation of the overall α-helical content compared with WT PrP (Fig. 2A). This is in agreement with NMR analysis of the segment 113–214 of Δ190–196 PrP (C1113), which contains the entire sequence of the structured domain and is an equivalent to the natural C1 fragment studied hereinafter. The large dispersion of amide chemical shifts observed in the 1H-15N HSQC spectrum of 15N13C-labeled mutant C1113 indicated that it maintained a globular core, in addition to its unstructured N-terminal region (Fig. 2B). Moreover, comparison with the spectra of Δ193–196 and Δ190–197 mutant PrPs previously obtained (23Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Chapuis J. Ciric D. Igel-Egalon A. Laude H. Béringue V. Rezaei H. Dron M. Generating bona fide mammalian prions with internal deletions.J. Virol. 2016; 90 (27226369): 6963-697510.1128/JVI.00555-16Crossref PubMed Scopus (9) Google Scholar, 24Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Igel-Egalon A. Barbereau C. Chapuis J. Ciric D. Laude H. Béringue V. Rezaei H. Dron M. A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity.Prion. 2017; 11 (28281924): 25-3010.1080/19336896.2016.1274851Crossref PubMed Scopus (3) Google Scholar) versus WT PrP showed that chemical shift perturbations followed a similar trend, confirming that the structure of the core domain of Δ190–196 C1113 is structurally close to those of other mutants (Fig. 2C). Last, analysis of Δ190–196 C111313Cα chemical shifts yielded the position of the three α-helices within residues 147–159, 175–189, and 203–230, showing that the topology is conserved with respect to WT PrP (Fig. 2C).Figure 1Δ190–196 PrP sequence. On the top is the schematic representation of mature ovine PrPC (positions 23–234). Secondary structures building the globular part of the protein, the two short beta strands forming a beta sheet, and the three α-helices are indicated. Post-translational modifications such as N-glycan chains (black dots), the disulfide bridge (S–S), and the GPI anchor are also shown. On the bottom, the amino acid sequence of α-helix H2 is highlighted in lavender, and the first residues of H3 are in purple. Residues 190–196 that were removed from the WT PrP are colored in red and replaced by a dotted line for the deletion mutant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Structure and stability of recombinant Δ190–196 PrP. A, comparative analysis of the secondary structures of WT PrP (blue) and Δ190–196 PrP (red) by CD. Far UV spectra indicate that the secondary structure of full-length WT PrP is essentially maintained in the mutant protein. B, NMR spectroscopy analysis of the C1-like segment (positions 113–234) of Δ190–196 mutant PrP. 2D 1H-15N HSQC spectrum of 250 μm recombinant 15N13C-labeled Δ190–196 C1113, acquired at a magnetic field of 18.8 T and a temperature of 298 K. C, combined amide chemical perturbations (obtained with weighing factors of 1 for 1H and 1/10 for 15N), measured for each nonproline residue with respect to WT PrP, are represented as superimposed bar diagrams. Perturbations are shown for Δ190–196 C1113 (red) and compared with those previously obtained for PrP Δ193–196 (cyan) and Δ190–197 (blue) (23Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Chapuis J. Ciric D. Igel-Egalon A. Laude H. Béringue V. Rezaei H. Dron M. Generating bona fide mammalian prions with internal deletions.J. Virol. 2016; 90 (27226369): 6963-697510.1128/JVI.00555-16Crossref PubMed Scopus (9) Google Scholar, 24Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Igel-Egalon A. Barbereau C. Chapuis J. Ciric D. Laude H. Béringue V. Rezaei H. Dron M. A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity.Prion. 2017; 11 (28281924): 25-3010.1080/19336896.2016.1274851Crossref PubMed Scopus (3) Google Scholar). The positions of the three α-helices, H1, H2, and H3, obtained by analysis of Δ190–196 C111313Cα chemical shifts by TALOS-N (71Shen Y. Bax A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.J. Biomol. NMR. 2013; 56 (23728592): 227-24110.1007/s10858-013-9741-yCrossref PubMed Scopus (725) Google Scholar) are shown at the top. D, comparison of stability between WT and Δ190–196 mutant PrP. Means melting temperatures (Tm) and standard deviations from five experiments were determined for full-length WT PrP (blue, 57.1 °C ± 0.9 °C) and Δ190–196 PrP (red, 49.9 °C ± 0.5 °C), for proteins resuspended in the same buffer conditions as for CD and NMR analysis (10 mm sodium acetate, pH 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The HTVTTTT deletion removed a histidine at position 190, which is the equivalent of His187 in human PrP (Fig. S1). The pH-dependent protonation of this histidine is thought to play an important role in the electrostatic network and the stability of the globular part of PrP (25Bae S.H. Legname G. Serban A. Prusiner S.B. Wright P.E. Dyson H.J. Prion proteins with pathogenic and protective mutations show similar structure and dynamics.Biochemistry. 2009; 48 (19618915): 8120-812810.1021/bi900923bCrossref PubMed Scopus (51) Google Scholar, 26Lee J. Chang I. Structural insight into conformational change in prion protein by breakage of electrostatic network around H187 due to its protonation.Sci. Rep. 2019; 9 (31848406): 1930510.1038/s41598-019-55808-1Crossref PubMed Scopus (8) Google Scholar). However, the deletion of the other residues might also impact the thermodynamic stability of the protein. We thus tested whether the deletion affected the stability using a thermal shift assay to determine the melting temperature of WT and mutant PrP. The melting temperature of Δ190–196 PrP (49.9 °C) was reduced by 7 °C compared with the WT PrP in sodium acetate buffer (10 mm, pH 5.0) (Fig. 2D). A marked reduction of 8 °C was also observed in these assays, using a different condition, sodium phosphate buffer (250 mm, pH 5.1), that increases the thermal stability of both WT and Δ190–196 recombinant PrPs (Fig. S2). Altogether these observations indicate that Δ190–196 mutant PrP conserves the overall structure of WT PrP but loses some stability. Ovine Δ190–196 PrP was stably transfected in RK13 cells to generate sublines and clones referred to as Δ190–196 Rov. Mutant PrP was efficiently expressed as a glycoprotein in Rov cells. Western blotting showed that unglycosylated forms of Δ190–196 PrPC were underrepresented compared with WT or Δ193–197 PrPs generated previously (23Munoz-Montesino C. Sizun C. Moudjou M. Herzog L. Reine F. Chapuis J. Ciric D. Igel-Egalon A. Laude H. Béringue V. Rezaei H. Dron M. Generating bona fide mammalian prions with internal deletions.J. Virol. 2016; 90 (27226369): 6963-697510.1128/JVI.00555-16Crossref PubMed Scopus (9) Google Scholar) (Fig. 3A). Deglycosylation by PNGase F treatment corroborated the high glycosylation level of Δ190–196 PrP and indicated that the mutant protein was smaller than WT PrPC by ∼1 kDa, as expected (Fig. 3B). Using an antibody with an epitope in the C-terminal part of PrP rather than in the N-terminal region allowed us to identify both the full-length protein and its natural C-terminal C1 fragment. PNGase treatment was required for accurate identification of PrPC and C1, because they are both highly glycosylated. The relative proportion of the full-length PrP versus the C1 fragment was roughly similar for the WT and the mutant protein (Fig. 3C). Immunofluorescence showed co-localization of Δ190–196 PrP with WGA, a lectin marker of plasma membrane glycoconjugates, indicating that the mutant protein was correctly addressed to the Δ190–196 Rov cell surface (Fig. 3D) as with the WT protein (27Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein.Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11259656): 4055-405910.1073/pnas.061337998Crossref PubMed Scopus (183) Google Scholar, 28Salamat M.K. Dron M. Chapuis J. Langevin C. Laude H. Prion propagation in cells expressing PrP glycosylation mutants.J. Virol. 2011; 85 (21248032): 3077-308510.1128/JVI.02257-10Crossref PubMed Scopus (31) Google Scholar). These observations indicate that Δ190–196 PrP has correct post-translational modifications and cell trafficking. The expression of Δ190–196 PrP was turned on by addition of doxycycline, and we followed the fate of the protein over cell passaging by Western blotting, checking for the appearance of PK-resistant forms. Although Δ190–196 PrP was sensitive to PK digestion during the first passages, a PK-resistant form systematically appeared, usually after the fourth or fifth passage (Fig. 4A). This protease-resistant form termed Δ190–196 PrPres persisted for >1 year of continuous culture (Fig. 5A). The electrophoretic profile of Δ190–196 PrPres was characterized by a large smear of glycosylated species and the presence of a well-individualized faint band migrating at 14 kDa (Fig. 4A). A second weaker band migrating at 15.5–16 kDa was detected upon overexposure of the blots or when enough material was loaded on the gel (Fig. 5A). Treatment with PNGase F allowed resolving the whole emerging PK-resistant species in two major bands: a main 14-kDa species that had the same size than the C1 fragment and a larger less represented peptide above the 15-kDa molecular mass marker that will be further referred to as 16-kDa PrPres (Fig. 4B). This indicated that the 14- and 16-kDa bands identified without PNGase treatment (Figs. 4A and 5A) were nonglycosylated native forms of Δ190–196 PrPres.Figure 5Persistence of the PK-resistant form in cultures, insolubility of PrPres, and reproducibility of spontaneous conversion. Immunoblots of PK-treated samples are shown. A, Δ190–196 PrPSc was produced persistently up to 1 year of continuous cell culture passage (1 per week). PrPres at 20, 30, and 52 weeks of culture are shown: 10 µg of total protein in 10 µl of cell lysate were digested and loaded (lanes 2–4). In lane 1 (P20+), the equivalent of 100 µg of protein from the passage at 20 weeks was loaded after PK digestion and concentration of insoluble material at 22,000 × g, to improve detection of the 14- and 16-kDa unglycosylated bands (Sha31 mAb). B, spontaneous emergence of Δ190–196 PrPres in three populations of Δ190–196 Rov cells obtained from independent transfections (lanes 1–3). C, individual clones isolated from two independent transfections. Five clones (lanes 1–5) with roughly similar Δ190–196 PrP expression levels (upper panel, 4F2 mAb) are shown. They produced spontaneously the PK-resistant form after eight passages of culture (lanes 1, 3, 4, and 5) with the exception of clone 12 (lane 2) (lower panel, Sha31 mAb).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The spontaneous emergence of PK-resistant Δ190–196 PrP was reproducible and occurred systematically from bulk cultures of Δ190–196 Rov obtained from three independent transfections (Fig. 5B). Individual Δ190–196 Rov clones obtained by limiting dilution spontaneously produced Δ190–196 PrPres, except for one clone, clone 12, despite expression of the mutant PrP to levels similar to those of other clones (Fig. 5C). This “resistant” clone was useful for the infection studies described in the following section, and its existence suggested that currently unrecognized cellular factors are key for the spontaneous generation of Δ190–196 PrPres. Δ190–196 Rov cells could be frozen and thawed, without affecting the generation of PK-resistant PrP species. This characteristic together with persistence in cell culture recalled that of prion-infected cells. We next examined whether the biochemical properties of Δ190–196 PrPSc resembled those of prions passaged in WT Rov cells. Δ190–196 Rov lysates were treated with increasing PK concentrations and analyzed by Western blotting. Δ190–196 PrPSc resisted to higher concentration of PK (Fig. 6, A and B) than 127S prions propagated in WT Rov (Fig. 6C). Δ190–196 PrPres was recovered by centrifugation at 20,000 × g after PK digestion, indicating that it was insoluble and aggregated (Fig. 5A). The aggregation size of Δ190–196 PrPSc was determined by sedimentation velocity. Δ190–196 PrPSc formed assemblies with a size in the range of PrPSc assemblies formed by subfibrillar prions (Fig. 6D) according to previous reports (29Tixador P. Herzog L. Reine F. Jaumain E. Chapuis J. Le Dur A. Laude H. Béringue V. The physical relationship between infectivity and prion protein aggregates is strain-dependent.PLoS Pathog. 2010; 6 (20419156): e100085910.1371/journal.ppat.1000859Crossref PubMed Scopus (124) Google Scholar, 30Igel-Egalon A. Moudjou M. Martin D. Busley A. Knäpple T. Herzog L. Reine F. Lepejova N. Richard C.A. Béringue V. Rezaei H. Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick.PLoS Pathog. 2017; 13 (28880932): e100655710.1371/journal.ppat.1006557Crossref PubMed Scopus (20) Google Scholar). 127S PrPSc assemblies from Rov cells had slightly larger assemblies with respect to size (Fig. 6D). Whether the difference is due to the number of PrP-mers composing the assemblies or to the density of their main core remains to be determined. To summarize, introduction of the 190–196 deletion in RK13 cells favored the spontaneous and persistent production of PK-resistant PrPSc species with an atypical electrophoretic pattern. Δ190–196 PrP spontaneously adopted a conformation that showed hallmarks of a prion: insolubility, aggregation, protease resistance and cell perpetuation. We thus called this e" @default.
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• W3048482863 date "2020-10-01" @default.
• W3048482863 modified "2023-10-16" @default.
• W3048482863 title "A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP" @default.
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• W3048482863 doi "https://doi.org/10.1074/jbc.ra120.014738" @default.
• W3048482863 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7549040" @default.
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