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Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms*

Open AccessPublished:October 26, 2012DOI:https://doi.org/10.1074/jbc.M112.398776
      The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrPC) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

      Introduction

      Prion disorders are dominant gain-of-function neurodegenerations whose pathogenesis is linked to misfolded forms of the cellular prion protein (PrPC),
      The abbreviations used are: PrP
      prion protein, CtmPrP, transmembrane PrPC
      Endo H
      endo-β-N-acetylglucosaminidase H
      ER
      endoplasmic reticulum
      GPI
      glycosylphosphatidylinositol
      PIPLC
      phosphatidylinositol phospholipase C
      PK
      proteinase K
      PNGase F
      peptide:N-glycosidase F
      PrPC
      cellular PrP
      PrPSc
      aggregated and protease-resistant β-sheet-enriched conformer of PrPC
      PrPDS
      PrP chain containing the combined M206S and M213S (M205S and M212S in mouse) substitution
      PDI
      protein disulfide isomerase
      βCOP
      β-coatomer protein.
      including the prion PrPSc and the neurotoxic CtmPrP (
      • Aguzzi A.
      • Calella A.M.
      Prions: protein aggregation and infectious diseases.
      ,
      • Hegde R.S.
      • Mastrianni J.A.
      • Scott M.R.
      • DeFea K.A.
      • Tremblay P.
      • Torchia M.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      A transmembrane form of the prion protein in neurodegenerative disease.
      ,
      • Hegde R.S.
      • Tremblay P.
      • Groth D.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      Transmissible and genetic prion diseases share a common pathway of neurodegeneration.
      ,
      • Prusiner S.B.
      Shattuck lecture: neurodegenerative diseases and prions.
      ). PrPSc is an aggregated and protease-resistant β-sheet-enriched conformer of PrPC, which self-perpetuates by the templating the conversion of cell surface PrPC (
      • Aguzzi A.
      • Calella A.M.
      Prions: protein aggregation and infectious diseases.
      ,
      • Prusiner S.B.
      Shattuck lecture: neurodegenerative diseases and prions.
      ). In contrast, CtmPrP is an intracellular transmembrane form generated at the ER with neurotoxic properties (
      • Aguzzi A.
      • Calella A.M.
      Prions: protein aggregation and infectious diseases.
      ,
      • Chakrabarti O.
      • Ashok A.
      • Hegde R.S.
      Prion protein biosynthesis and its emerging role in neurodegeneration.
      ,
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ). Despite that CtmPrP formation was associated with features of the ER translocation process several pathogenic mutations in the C-terminal domain such as H187R and E200K enhance its levels, suggesting a yet unexplored in vivo interplay between folding and the accumulation and action of this neurotoxic form (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Kim S.J.
      • Hegde R.S.
      Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel.
      ,
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      • Shi Q.
      • Xu K.
      • Gao C.
      • Chen C.
      • Li X.L.
      • Wang G.R.
      • Tian C.
      • Han J.
      • Dong X.P.
      Familial CJD-associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.
      ).
      Since the enunciation of the prion hypothesis, research has focused on the mechanism by which a native PrPC structure reorganizes and acquires self-propagative features like those of PrPSc (
      • Gasset M.
      • Baldwin M.A.
      • Lloyd D.H.
      • Gabriel J.M.
      • Holtzman D.M.
      • Cohen F.
      • Fletterick R.
      • Prusiner S.B.
      Predicted α-helical regions of the prion protein when synthesized as peptides form amyloid.
      ,
      • Makarava N.
      • Kovacs G.G.
      • Savtchenko R.
      • Alexeeva I.
      • Budka H.
      • Rohwer R.G.
      • Baskakov I.V.
      Genesis of mammalian prions: from noninfectious amyloid fibrils to a transmissible prion disease.
      ,
      • Pan K.M.
      • Baldwin M.
      • Nguyen J.
      • Gasset M.
      • Serban A.
      • Groth D.
      • Mehlhorn I.
      • Huang Z.
      • Fletterick R.J.
      • Cohen F.E.
      Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins.
      ). The PrPC native state was assigned to the fold adopted by the chain lacking the signal sequences and containing the disulfide bond and used as reference for testing the effect of pathogenic mutations and its conversion into active prions (
      • Makarava N.
      • Kovacs G.G.
      • Savtchenko R.
      • Alexeeva I.
      • Budka H.
      • Rohwer R.G.
      • Baskakov I.V.
      Genesis of mammalian prions: from noninfectious amyloid fibrils to a transmissible prion disease.
      ,
      • Apetri A.C.
      • Surewicz K.
      • Surewicz W.K.
      The effect of disease-associated mutations on the folding pathway of human prion protein.
      ,
      • Liemann S.
      • Glockshuber R.
      Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein.
      ,
      • Meli M.
      • Gasset M.
      • Colombo G.
      Dynamic diagnosis of familial prion diseases supports the β2–α2 loop as a universal interference target.
      ,
      • Riek R.
      • Hornemann S.
      • Wider G.
      • Billeter M.
      • Glockshuber R.
      • Wüthrich K.
      NMR structure of the mouse prion protein domain PrP(121–231).
      ,
      • van der Kamp M.W.
      • Daggett V.
      Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding.
      ). However, the in vivo folding of proteins segregating into the secretory route such as PrP is a complex process participated by the ER folding machinery. This machinery coordinates processing (signal sequences removal, addition of covalent modifications, binding of cofactors, etc.), avoids undesired aggregations, and permits the acquisition of correct structure. This global process involves multiple transient protein-protein interactions with the nascent chains that can sense alterations resulting from environmental changes to the presence of mutations (
      • Anelli T.
      • Sitia R.
      Protein quality control in the early secretory pathway.
      ,
      • Hartl F.U.
      • Hayer-Hartl M.
      Converging concepts of protein folding in vitro in vivo.
      ,
      • Kim P.S.
      • Arvan P.
      Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones.
      ). Any variation in the sequence of events can impact the final product, as for the doses of secretory and transmembrane PrP forms, and its fate (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Kim S.J.
      • Hegde R.S.
      Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel.
      ,
      • Ashok A.
      • Hegde R.S.
      Selective processing and metabolism of disease-causing mutant prion proteins.
      ,
      • Campana V.
      • Sarnataro D.
      • Fasano C.
      • Casanova P.
      • Paladino S.
      • Zurzolo C.
      Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum.
      ,
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Drisaldi B.
      • Stewart R.S.
      • Adles C.
      • Stewart L.R.
      • Quaglio E.
      • Biasini E.
      • Fioriti L.
      • Chiesa R.
      • Harris D.A.
      Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.
      ,
      • Ivanova L.
      • Barmada S.
      • Kummer T.
      • Harris D.A.
      Mutant prion proteins are partially retained in the endoplasmic reticulum.
      ,
      • Kiachopoulos S.
      • Bracher A.
      • Winklhofer K.F.
      • Tatzelt J.
      Pathogenic mutations located in the hydrophobic core of the prion protein interfere with folding and attachment of the glycosylphosphatidylinositol anchor.
      ,
      • Lorenz H.
      • Windl O.
      • Kretzschmar H.A.
      Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases.
      ,
      • Orsi A.
      • Sitia R.
      Interplays between covalent modifications in the endoplasmic reticulum increase conformational diversity in nascent prion protein.
      ,
      • Rosenmann H.
      • Talmor G.
      • Halimi M.
      • Yanai A.
      • Gabizon R.
      • Meiner Z.
      Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrPC.
      ,
      • Schiff E.
      • Campana V.
      • Tivodar S.
      • Lebreton S.
      • Gousset K.
      • Zurzolo C.
      Coexpression of wild-type and mutant prion proteins alters their cellular localization and partitioning into detergent-resistant membranes.
      ,
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Vetrugno V.
      • Malchow M.
      • Liu Q.
      • Marziali G.
      • Battistini A.
      • Pocchiari M.
      Expression of wild-type and V210I mutant prion protein in human neuroblastoma cells.
      ,
      • Yanai A.
      • Meiner Z.
      • Gahali I.
      • Gabizon R.
      • Taraboulos A.
      Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.
      ,
      • Yin S.
      • Pham N.
      • Yu S.
      • Li C.
      • Wong P.
      • Chang B.
      • Kang S.C.
      • Biasini E.
      • Tien P.
      • Harris D.A.
      • Sy M.S.
      Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans.
      ).
      Metabolic studies addressing the effect of pathogenic mutations in the C-terminal domain of PrP as disease predisposition factors have reported a wide range of alterations in processing, trafficking, aggregation, accumulation, and toxicity which varied among experimental setups, as the cell line used and the background expression of wild-type (WT) PrPC (
      • Ashok A.
      • Hegde R.S.
      Selective processing and metabolism of disease-causing mutant prion proteins.
      ,
      • Campana V.
      • Sarnataro D.
      • Fasano C.
      • Casanova P.
      • Paladino S.
      • Zurzolo C.
      Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum.
      ,
      • Drisaldi B.
      • Stewart R.S.
      • Adles C.
      • Stewart L.R.
      • Quaglio E.
      • Biasini E.
      • Fioriti L.
      • Chiesa R.
      • Harris D.A.
      Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.
      ,
      • Ivanova L.
      • Barmada S.
      • Kummer T.
      • Harris D.A.
      Mutant prion proteins are partially retained in the endoplasmic reticulum.
      ,
      • Kiachopoulos S.
      • Bracher A.
      • Winklhofer K.F.
      • Tatzelt J.
      Pathogenic mutations located in the hydrophobic core of the prion protein interfere with folding and attachment of the glycosylphosphatidylinositol anchor.
      ,
      • Lorenz H.
      • Windl O.
      • Kretzschmar H.A.
      Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases.
      ,
      • Rosenmann H.
      • Talmor G.
      • Halimi M.
      • Yanai A.
      • Gabizon R.
      • Meiner Z.
      Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrPC.
      ,
      • Schiff E.
      • Campana V.
      • Tivodar S.
      • Lebreton S.
      • Gousset K.
      • Zurzolo C.
      Coexpression of wild-type and mutant prion proteins alters their cellular localization and partitioning into detergent-resistant membranes.
      ,
      • Vetrugno V.
      • Malchow M.
      • Liu Q.
      • Marziali G.
      • Battistini A.
      • Pocchiari M.
      Expression of wild-type and V210I mutant prion protein in human neuroblastoma cells.
      ,
      • Yin S.
      • Pham N.
      • Yu S.
      • Li C.
      • Wong P.
      • Chang B.
      • Kang S.C.
      • Biasini E.
      • Tien P.
      • Harris D.A.
      • Sy M.S.
      Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans.
      ). These aberrancies contrast with structural reports in which the same pathogenic mutations do not impede the correct in vitro folding, but variably modify the stability, dynamics, and surface reactivity of the native state (
      • Apetri A.C.
      • Surewicz K.
      • Surewicz W.K.
      The effect of disease-associated mutations on the folding pathway of human prion protein.
      ,
      • Liemann S.
      • Glockshuber R.
      Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein.
      ,
      • Meli M.
      • Gasset M.
      • Colombo G.
      Dynamic diagnosis of familial prion diseases supports the β2–α2 loop as a universal interference target.
      ,
      • Riek R.
      • Hornemann S.
      • Wider G.
      • Billeter M.
      • Glockshuber R.
      • Wüthrich K.
      NMR structure of the mouse prion protein domain PrP(121–231).
      ,
      • van der Kamp M.W.
      • Daggett V.
      Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding.
      ,
      • Calzolai L.
      • Lysek D.A.
      • Guntert P.
      • von Schroetter C.
      • Riek R.
      • Zahn R.
      • Wüthrich K.
      NMR structures of three single-residue variants of the human prion protein.
      ,
      • Zahn R.
      • Liu A.
      • Lührs 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.
      ). Indeed, aging factors such as oxidative modifications and exhaustion of the ER folding machinery which are not considered in structural studies may play fundamental roles in the formation of pathogenic PrP.
      Of the different mutations in the globular domain experimentally tested, substitutions of conserved methionines in α-helix 3 (hitherto PrPα3M) provoked the largest α-fold destabilization (
      • Lisa S.
      • Meli M.
      • Cabello G.
      • Gabizon R.
      • Colombo G.
      • Gasset M.
      The structural intolerance of the PrP α-fold for polar substitution of the helix-3 methionines.
      ). In particular, singly or combined M206S and M213S replacements in rHaPrP(23–231) yielded extremely labile folds with enhanced aggregation capacity (
      • Lisa S.
      • Meli M.
      • Cabello G.
      • Gabizon R.
      • Colombo G.
      • Gasset M.
      The structural intolerance of the PrP α-fold for polar substitution of the helix-3 methionines.
      ). These mutations also mimicked the flexibility distortions impinged by sulfoxidation of such methionines found in the PrP chains in the conversion pathway (
      • Apetri A.C.
      • Surewicz K.
      • Surewicz W.K.
      The effect of disease-associated mutations on the folding pathway of human prion protein.
      ,
      • Liemann S.
      • Glockshuber R.
      Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein.
      ,
      • Meli M.
      • Gasset M.
      • Colombo G.
      Dynamic diagnosis of familial prion diseases supports the β2–α2 loop as a universal interference target.
      ,
      • Lisa S.
      • Meli M.
      • Cabello G.
      • Gabizon R.
      • Colombo G.
      • Gasset M.
      The structural intolerance of the PrP α-fold for polar substitution of the helix-3 methionines.
      ,
      • Canello T.
      • Engelstein R.
      • Moshel O.
      • Xanthopoulos K.
      • Juanes M.E.
      • Langeveld J.
      • Sklaviadis T.
      • Gasset M.
      • Gabizon R.
      Methionine sulfoxides on PrPSc: a prion-specific covalent signature.
      ,
      • Canello T.
      • Frid K.
      • Gabizon R.
      • Lisa S.
      • Friedler A.
      • Moskovitz J.
      • Gasset M.
      Oxidation of helix-3 methionines precedes the formation of PK-resistant PrP.
      ,
      • Colombo G.
      • Meli M.
      • Morra G.
      • Gabizon R.
      • Gasset M.
      Methionine sulfoxides on prion protein helix-3 switch on the α-fold destabilization required for conversion.
      ). Despite these interesting results, the effect of these substitutions had not been addressed in living systems.
      Here, we have used various cultured cells expressing PrPα3M mutants to investigate and model the role of in vivo folding in the synthesis and accumulation of PrP forms. Unexpectedly, we found that the PrPα3M expression is highly toxic and that such toxicity relates to the exclusive formation of CtmPrP due to impeded disulfide bond formation.

      EXPERIMENTAL PROCEDURES

      Plasmid Construction and PrP Mutant Preparation

      The pcDNA3.1-MoPrP(1–254, 3F4-tagged), pcDNA4.1-HaPrP(1–254) (
      • Juanes M.E.
      • Elvira G.
      • García-Grande A.
      • Calero M.
      • Gasset M.
      Biosynthesis of prion protein nucleocytoplasmic isoforms by alternative initiation of translation.
      ,
      • Rane N.S.
      • Yonkovich J.L.
      • Hegde R.S.
      Protection from cytosolic prion protein toxicity by modulation of protein translocation.
      ), and pHaPrP-YFP (
      • Domingo B.
      • Gasset M.
      • Durán-Prado M.
      • Castaño J.P.
      • Serrano A.
      • Fischer T.
      • Llopis J.
      Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.
      ) were used as templates for the generation of MoPrP M134S, MoPrP M154S, MoPrP M205S, MoPrP M212S, MoPrP M205S,M212S, HaPrP M134S, HaPrP M154S, HaPrP M206S, HaPrP M213S, HaPrP M206S,M213S, HaPrP M213L, HaPrP A117V, HaPrP C214A, HaPrP G123P. and HaPrP G123P,M206S,M213S mutants. Site-directed mutagenesis was carried out using QuikChange protocols with the oligonucleotides summarized in Table 1.
      TABLE 1Primers used for mutagenesis
      MutationTemplatePrimer (forward, 5′-3′)
      M134SMoPrPATGCTGGGGAGCGCCAGTAGCAGGCCCATGATC
      M154SMoPrPTACTACCGTGAAAACAGTTACCGCTACCCTAAC
      M205SMoPrPACCGATGTGAAGATGAGTGAGCGCGTGGTGGAG
      M212SMoPrPCGCGTGGTGGAGCAGAGTTGCGTCACCCAGTAC
      M205S,M212SMoPrP M205SCGCGTGGTGGAGCAGAGTTGCGTCACCCAGTAC
      M134SHaPrPCTGGGGAGTGCCTCCTCTAGACCCATG
      HaPrP-YFP
      M154SHaPrPCTACCGTGAAAACTCCAATCGATACCC
      HaPrP-YFP
      M206SHaPrPGACATCAAGATATCCGAGCGCGTGG
      HaPrP-YFP
      M213SHaPrPGTGGTGGAGCAGTCCTGTACCACCCAG
      HaPrP-YFP
      M206S,M213SHaPrP M206SGTGGTGGAGCAGTCCTGTACCACCCAG
      HaPrP-YFP-M206S
      M213LHaPrPGTGGTGGAGCAGCTCTGTACCACCCAG
      HaPrP-YFP
      A117VHaPrPGCCGGCGCTGCTGTGGCAGGGGCC
      HaPrP-YFP
      C214AHaPrPGAGCGCGTGGTGGAGCAGATGGCTACCACCCAGTATCAG
      HaPrP-YFP
      G123PHaPrPGCCGTGGTGCCGGGCCTTGGTG
      HaPrP-YFP
      HaPrPDS
      HaPrP- YFPDS

      Cell Culture, Transfections, and Viability Assays

      The cell lines HpL 3.14 (PrP−/−, mouse hypothalamic), GpL (PrP−/− mouse glial), N2a (mouse neuroblastoma), and CHO (Chinese hamster ovary) cells were kept in Opti-MEM containing 10% fetal bovine serum and penicillin/streptomycin (
      • Juanes M.E.
      • Elvira G.
      • García-Grande A.
      • Calero M.
      • Gasset M.
      Biosynthesis of prion protein nucleocytoplasmic isoforms by alternative initiation of translation.
      ,
      • Kuwahara C.
      • Takeuchi A.M.
      • Nishimura T.
      • Haraguchi K.
      • Kubosaki A.
      • Matsumoto Y.
      • Saeki K.
      • Matsumoto Y.
      • Yokoyama T.
      • Itohara S.
      • Onodera T.
      Prions prevent neuronal cell-line death.
      ,
      • Nishimura T.
      • Sakudo A.
      • Xue G.
      • Ikuta K.
      • Yukawa M.
      • Sugiura K.
      • Onodera T.
      Establishment of a new glial cell line from hippocampus of prion protein gene-deficient mice.
      ,
      • Birkett C.R.
      • Hennion R.M.
      • Bembridge D.A.
      • Clarke M.C.
      • Chree A.
      • Bruce M.E.
      • Bostock C.J.
      Scrapie strains maintain biological phenotypes on propagation in a cell line in culture.
      ). Transient transfections with the different PrP-coding plasmid, pEGFP-rab9 (positive control for transfection), and pcDNA3.1/pcDNA4.1 (mock control) were performed using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Typically, 24 h after transfection the medium was exchanged and allowed for other 24 h before analysis. For viability assays, cells were harvested using trypsin/EDTA in PBS followed by centrifugation and stained with 0.4% trypan blue for 5 min at room temperature. Total and viable cells were determined using the TC10 automated cell counter (Bio-Rad) using duplicate independent readings of experiments performed in duplicates. Displayed data are the mean ± S.D. of three independent experiments. Statistical analyses were performed using the t test, with significance set to p < 0.05. For other analysis, cells were harvested by either in situ lysis in cold radioimmune precipitation assay buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm EDTA, 0.5% Triton X-100, 0.5% deoxycholate) or by detachment with PBS containing 10 mm EDTA.

      Immunoblot Analysis

      Samples were diluted in Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, and 0.125 m Tris-HCl), heated at 95 °C for 5 min, and then resolved by SDS-PAGE (12% gel). Proteins were electrophoretically transferred onto either PVDF or nitrocellulose membranes, and the membranes were blocked for 1 h in 5% (w/v) nonfat dried skimmed milk powder in Tris-buffered saline containing 0.05% Tween 20. After incubation with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies, signals were revealed using enhanced chemiluminescence (Amersham Biosciences) and visualized using the ChemiDoc detector (Bio-Rad). The primary antibodies were: mouse anti-PrP 3F4(109–112) HaPrP region, 1:20,000; Signet), 6D11 (1:10,000; 90–100 region; Covance); anti-β-actin (1:5000; Sigma-Aldrich), anti-CHOP (1:1000; Santa Cruz Biotechnology), anti-GRP78 (1:1000; Santa Cruz Biotechnology), anti-protein disulfide isomerase (1:1000; Abcam), and anti-fluorescein (1:10,000; Invitrogen). The secondary antibodies were: goat anti-mouse HRP (1:5000; Sigma-Aldrich), goat anti-rabbit HRP (1:5000; Chemicon). Quantitative densitometry of protein bands was performed using Quantity One software (Bio-Rad).

      Expression Analysis, Detergent Solubility Assay, and Proteinase K Treatments

      Cell lysates were cleared by a centrifugation of 5 min at 500 × g, supplemented with 0.5 mm Pefabloc, and then precipitated with 5 volumes of methanol at −20 °C. Samples were centrifuged at 10,000 × g for 30 min, and the pellets were redissolved in TNE buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA). For expression studies, aliquots of the TNE-resuspended samples were diluted with Laemmli buffer and analyzed by immunoblotting. For solubility assay, the cleared cell lysates were supplemented with sarkosyl to 1% and centrifuged for 1 h at 100,000 × g, 4 °C, in a Beckman OptimaTM Max centrifuge. Soluble fractions (supernatant) were precipitated with methanol and then together with the insoluble fractions (pellet) were analyzed by immunoblotting. For protease digestions, aliquots of cleared lysates prepared in the absence of Pefabloc were incubated for 30 min at 37 °C with 20 μg/ml proteinase K (PK; Promega); the proteolysis was stopped by addition of 5 mm protease inhibitor PMSF. Samples were precipitated with methanol and analyzed by immunoblotting. For protease protection assays, detached cells were homogenized in 10 mm Tris-HCl, pH 7.4, 0.1 m sodium acetate, 2 mm MgCl2 and centrifuged at 1000 × g at 4 °C for 10 min. The supernatants were then centrifuged for 1 h at 100,000 × g at 4 °C, and the resulting pellets (microsomes) were resuspended in 50 mm Hepes, pH 7.4, 0.1 m sodium acetate, 2 mm MgCl2, 0.25 m sucrose. Microsome suspensions were split in three equal parts and incubated in the absence and presence of PK (5 μg/ml), both with and without 0.5% Triton X-100, for 30 min at 25 °C. Reactions were stopped by adding 0.5 mm PMSF and analyzed by immunoblotting.

      Post-translational Covalent Modification Analysis: Glycosylation, GPI Addition, and Disulfide Bonding

      Enzymatic digestions with PNGase F (New England Biolabs), Endo H (New England Biolabs), and phosphatidylinositol phospholipase C (PIPLC) (Sigma-Aldrich) were performed for 1 h at 37 °C on the methanol-precipitated cell lysates, following the manufacturer's instructions. After digestion, reactions were stopped by the addition of Laemmli buffer, and PrP was analyzed by immunoblotting using 3F4 antibody. For analysis of disulfide bonds, cells were lysed in 50 mm Tris-HCl, pH 8, 1% SDS, cleared by a slow speed centrifugation, and then supplemented with or without 200 mm DTT. Both reduced and nonreduced lysates were then incubated with 100 mm Oregon Green 488 iodoacetamide (Invitrogen) for 15 min, diluted with 10 volumes of radioimmune precipitation assay buffer, and incubated with protein A-Sepharose beads (GE Healthcare) for 60 min at 4 °C. After centrifugation, supernatants were incubated with mAb 3F4 for 10 h at 4 °C. Protein A-Sepharose beads were then added, and after a 90-min incubation at 4 °C, the protein-antibody complexes bound to protein A-agarose were sedimented by centrifugation. Pellets were washed with a buffer containing 150 mm NaCl, 10 mm Tris-HCl, pH 7.8, 0.1% sarkosyl, and 0.1% Pefabloc, and bound proteins were eluted by boiling in SDS-sample buffer. Precipitates were analyzed by SDS-PAGE and developed with goat anti-fluorescein/Oregon Green Ab (Invitrogen).

      Fluorescence Microscopy Imaging

      Cells were seeded on to poly-l-lysine-coated glass coverslips, transfected with the plasmids coding for HaPrP WT, HaPrP-YFP WT, and their mutants, and grown for 36 h to 60% confluence. For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde in PBS containing 5% sucrose for 10 min at room temperature and washed three times with PBS. Cells were permeabilized and blocked in PBS containing 0.5% saponin, 0.1% Triton X-100, and 2% bovine serum albumin for 10 min at room temperature. Cells were incubated with anti-PrP mAb 3F4 (1:600) and with anti-β coatomer protein (1:600) for 1 h at room temperature. After washing with blocking buffer, samples were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:800), Alexa Fluor 647-conjugated anti-rabbit IgG (1:800), and Hoechst 33342 (10 μg/ml) in for 30 min at room temperature. Once washed, the coverslips were mounted on glass slides with ProLong Gold antifade reagent (Invitrogen). Images were captured with a confocal microscope (Leica TCS-SP-AOBS-UV) as described (
      • Juanes M.E.
      • Elvira G.
      • García-Grande A.
      • Calero M.
      • Gasset M.
      Biosynthesis of prion protein nucleocytoplasmic isoforms by alternative initiation of translation.
      ). For living cell analysis, imaging was performed as described previously (
      • Domingo B.
      • Gasset M.
      • Durán-Prado M.
      • Castaño J.P.
      • Serrano A.
      • Fischer T.
      • Llopis J.
      Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.
      ).

      RESULTS

      PrPα3M Mutants Are Highly Toxic in Cultured Cells

      To determine the role of α3M mutations on PrP folding in living cells we generated Met to Ser substitutions in the α-helix 3 both in HaPrP (M206S, M213S, DS) and MoPrP (M205S, M212S, DS) (Fig. 1A). We also generated control substitutions at other conserved methionines (M134S and M154S), a substitution that preserves both the conformation and stability of the recombinant chain (M213L) and a pathogenic mutant known to alter in vivo PrP folding (A117V) (
      • Hegde R.S.
      • Mastrianni J.A.
      • Scott M.R.
      • DeFea K.A.
      • Tremblay P.
      • Torchia M.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      A transmembrane form of the prion protein in neurodegenerative disease.
      ,
      • Hegde R.S.
      • Tremblay P.
      • Groth D.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      Transmissible and genetic prion diseases share a common pathway of neurodegeneration.
      ,
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Lisa S.
      • Meli M.
      • Cabello G.
      • Gabizon R.
      • Colombo G.
      • Gasset M.
      The structural intolerance of the PrP α-fold for polar substitution of the helix-3 methionines.
      ) (Fig. 1A). We expressed all of these constructs in PrP−/− cells (HpL and GpL) to avoid interference from endogenous WT PrP chains (Fig. 1A). Transient expression of HaPrP constructs revealed specific cytotoxicity of PrPα3M mutants in both cell types (Fig. 1B). HaPrP WT and the control M134S, M154S, and M213L mutations all had no effect on cell viability, whereas HaPrPα3M mutants caused approximately 40% cell death in both HpL and GpL cells upon transient transfection (Fig. 1B). Interestingly, HaPrP A117V also induced 30% cell death, suggesting possible similarities in the pathogenic process (Fig. 1B). PrPα3M mutations generated as HaPrP-YFP fusions (
      • Domingo B.
      • Gasset M.
      • Durán-Prado M.
      • Castaño J.P.
      • Serrano A.
      • Fischer T.
      • Llopis J.
      Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.
      ) showed the same cell loss as those on HaPrP background, arguing that YFP does not interfere in the lethal process (Fig. 1B). Next, we tested the toxicity of PrPα3M mutants using MoPrP sequence. As with the HaPrP constructs, we found that the three PrPα3M mutants induced approximately 40–50% cell death (Fig. 1C). Thus, the scaffold sequence had no effect on PrPα3M toxicity, further supporting the crucial role of these conserved residues on the in vivo PrP folding. Importantly, toxicity increases with the dose of PrPα3M expression, as shown for variation of cell death relative to the normalized HaPrPDS signal as a function of the time after transfection (Fig. 1D).
      Figure thumbnail gr1
      FIGURE 1The expression of the PrP α-fold impairing α3M mutations is highly toxic for mammalian PrP−/− cells. A, schematic represents PrP mutants used in this study. Red rectangles, translocation-controlling regions (N-terminal signal sequence and hydrophobic region); green rectangles, secondary structure elements of the globular fold (α, α-helix; β, β-strand); purple line, disulfide bond; orange arrow, site of GPI anchor addition; yellow rectangle, insertion site of YFP for fluorescence fusions. B, transient expression of HaPrP WT and of its mutants on HpL (blue) and GpL (orange) impacts viability. C, transient expression of WT and mutant MoPrP affects HpL (blue) and GpL (orange) viability. D, variation of cell death was normalized to 3F4 signal with the post-transfection time for HaPrP WT and HaPrPDS. Percentage of cell death was obtained from viability assays, and 3F4 signal was obtained from immunoblots after normalization to the signal of β-actin band. E, co-expression of WT PrP and PrPα3M mutants preserved mutant toxicity. F, PrPα3M mutants retain the toxicity in both N2a and nonneural CHO cells. Displayed data are the mean ± S.D. (error bars) of at least three independent experiments performed in duplicate. Statistical analyses were performed using the t test, with significance set to p < 0.05.
      Having seen that PrPα3M mutants are highly toxic to culture cells not expressing an endogenous PrP, we wanted to study whether expression of WT PrP affects this phenotype. We co-expressed HaPrP and MoPrP bearing the double mutant (PrPDS) with the corresponding WT PrP in a 1:1 ratio in GpL and HpL cells to determine whether this could ameliorate the toxic effect. Fig. 1E shows that PrP WT had no or little effect on PrPDS toxicity in this setup. Next, we transiently expressed both HaPrPDS and MoPrPDS in N2a cells, which express MoPrPC, and in nonneuronal CHO cells, which lack detectable PrP (Fig. 1F). Again, there was substantial toxicity in tested cells, which was apparently independent of endogenous WT PrP expression and cell type used. Altogether, these results indicate that PrPα3M mutants exert a very high level of cell toxicity upon transient expression which is not influenced by cell type and co-expression of WT PrP.

      PrPα3M Mutants Are Processed Abnormally during Biogenesis

      After determining that PrPα3M mutants are highly toxic, we set out to characterize the molecular basis of such toxicity. Expression of WT and the control M134S and M154S mutants produced the classical 24–38-kDa banding pattern for both HaPrP and MoPrP constructs and in both PrP−/− cell lines (Fig. 2A). However, expression of PrPα3M mutants yielded single bands of approximately 30 kDa (Fig. 2A). This feature has been described previously for PrP pathogenic mutants with immature glycosylation (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Ashok A.
      • Hegde R.S.
      Selective processing and metabolism of disease-causing mutant prion proteins.
      ,
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Kiachopoulos S.
      • Bracher A.
      • Winklhofer K.F.
      • Tatzelt J.
      Pathogenic mutations located in the hydrophobic core of the prion protein interfere with folding and attachment of the glycosylphosphatidylinositol anchor.
      ,
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Yanai A.
      • Meiner Z.
      • Gahali I.
      • Gabizon R.
      • Taraboulos A.
      Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.
      ,
      • Stewart R.S.
      • Drisaldi B.
      • Harris D.A.
      A transmembrane form of the prion protein contains an uncleaved signal peptide and is retained in the endoplasmic reticulum.
      ,
      • Stewart R.S.
      • Harris D.A.
      A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons.
      ). This stimulated us to study the glycosylation of PrPα3M mutants, focusing on HaPrPDS as a model. Indeed, whereas WT HaPrP was sensitive to PNGase F and resistant to Endo H digestions, the glycan attached to HaPrPDS was sensitive to both PNGase F and Endo H digestion (Fig. 2B). These results indicate an immature glycosylation of the PrPDS mutant. To test for the presence of a GPI anchor, the PNGase F-deglycosylated products were further digested with PIPLC. The upward shift in the bands of WT HaPrP and HaPrPDS after PIPLC treatment indicates the removal of the hydrophobic moiety and supported the presence of the GPI anchor in both chains (Fig. 2B). Also, the size similarity of WT HaPrP and HaPrPDS bands upon combined PNGase F and PIPLC digestion indicates that in both PrP chains the N-terminal signal sequence has been removed. Taken together, the post-translational processing of HaPrPDS, representative here of PrP3αM mutants, indicates removal of the N-terminal signal sequence, immature glycosylation, and the presence of a GPI anchor.
      Figure thumbnail gr2
      FIGURE 2Molecular features of PrP mutants in mammalian cells. A, transient expression of HaPrP and MoPrP WT and mutants in HpL and GpL cells. The signal corresponding to β-actin is displayed at the bottom of each membrane. B, PNGase F, Endo H, and PIPLC digestions of HaPrP WT and HaPrPDS expressed in HpL cells. C, differences in the detergent solubility of HpL-expressed HaPrP WT and HaPrPDS determined by partitioning between the soluble and pellet fraction of an ultracentrifugation. D, protease sensitivity at 37 and 4 °C of HpL-expressed HaPrP WT and HaPrPDS probed by PK digestion. E, PK protection of PrP chains in intact (M) and lysed (H) microsomes isolated from HpL-expressed HaPrP WT, M134S, M154S, DS, C214A, M213L, and A117V, this last taken as positive control for CtmPrP topology. F, transient expression of HaPrPG123P and HaPrPG123PDS in HpL cells. The signal corresponding to β-actin is displayed at the bottom. G, PK protection of PrP chains in intact (M) and lysed (H) microsomes isolated from HpL transiently expressing HaPrPG123P and HaPrPG123PDS mutants. PrP immunoblotting was performed with mAb 3F4.

      PrPα3M Mutants Accumulate as CtmPrP

      The above biochemical features were reminiscent of previously described de novo formation of either PrPSc-like or CtmPrP forms (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Juanes M.E.
      • Elvira G.
      • García-Grande A.
      • Calero M.
      • Gasset M.
      Biosynthesis of prion protein nucleocytoplasmic isoforms by alternative initiation of translation.
      ,
      • Rane N.S.
      • Yonkovich J.L.
      • Hegde R.S.
      Protection from cytosolic prion protein toxicity by modulation of protein translocation.
      ,
      • Resenberger U.K.
      • Winklhofer K.F.
      • Tatzelt J.
      Neuroprotective and neurotoxic signaling by the prion protein.
      ). To assess the conformation adopted by PrPα3M mutants, we analyzed the detergent solubility and the resistance to proteases of HaPrPDS (
      • Aguzzi A.
      • Calella A.M.
      Prions: protein aggregation and infectious diseases.
      ,
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ). Fig. 2C shows that WT HaPrP proved completely soluble in detergents whereas HaPrPDS partitioned almost entirely into the insoluble fraction. Nonetheless, both proteins were fully digested by PK under harsh (37 °C) and mild (4 °C) digestion conditions, supporting the absence of prototypic PrPSc characteristics (Fig. 2D).
      Once discarded the generation of bona fide and PK-resistant PrPSc, we next analyzed the membrane topology of HaPrPα3M, using HaPrPA117V as control for acquiring CtmPrP topology. For this, we purified microsomes from transfected cells to carry out protease protection assays (Fig. 2E). HaPrP WT and its M134S, M154S, and M213L mutants were fully protected under mild conditions (PK digestion without detergent) but completely digested in the presence of detergents, supporting the production of the secretory PrP form. In contrast, under mild digestion conditions HaPrP A117V revealed a protease-protected fragment of approximately 21 kDa, corresponding to the ER-localized C-terminal domain characteristic of CtmPrP (
      • Kim S.J.
      • Hegde R.S.
      Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel.
      ). Digestion of microsomes from cells expressing HaPrPM206S, HaPrP213S, and HaPrPDS yielded the same 21-kDa fragment as identified for HaPrP A117V. In summary, these results indicate that HaPrPα3M folding results in acquisition of a transmembrane CtmPrP topology.

      PrPα3M Mutants Are Retained Intracellularly and Exert ER Stress

      Because CtmPrP is mainly an intracellular conformer and has been proposed to elicit an ER stress response, we then tested these properties for HaPrPα3M (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Wang X.
      • Shi Q.
      • Xu K.
      • Gao C.
      • Chen C.
      • Li X.L.
      • Wang G.R.
      • Tian C.
      • Han J.
      • Dong X.P.
      Familial CJD-associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.
      ,
      • Harris D.A.
      Trafficking, turnover and membrane topology of PrP.
      ). First, we performed indirect fluorescence microscopy studies to address the subcellular location upon transient transfection into HpL cells. As expected, WT HaPrP and its control M134S and M213L mutants were located along the secretory pathway in route to the plasma membrane (Fig. 3, A–C). In contrast, HaPrPα3M mutants were found mostly intracellularly, with a punctuate distribution overlapping partially the Golgi (co-staining with the marker βCOP) and ER (co-staining with the marker PDI; data not shown) membranes (Fig. 3, E–G). We also analyzed the distribution of the corresponding YFP fusion constructs. Again, WT HaPrP-YFP, HaPrPM134S-YFP, and HaPrPM213L-YFP were found mainly at the plasma membrane with some Golgi staining indicating a normal behavior (Fig. 3, J–L). However, the HaPrP HaPrPα3M-YFP fusions, which retained toxicity (see Fig. 1B), accumulated intracellularly displaying an excess of intracellular vesicle structures containing PrP which most likely represent ER compartments (Fig. 3, N–P). Thus, the abnormal intracellular distribution of PrPα3M mutants observed here by microscopy is in line with our biochemical analysis showing immature glycosylation, tendency to aggregate, and CtmPrP topology.
      Figure thumbnail gr3
      FIGURE 3PrPα3M accumulates intracellularly. Subcellular distribution of WT HaPrP (A and J) and of its M134S (B and K), M213L (C and L), G123P (D and M), M206S (E and N), M213S (F and O), DS (G and P), C214A (H and Q), and G123PDS (I and R) mutants expressed in HpL cells is shown. Panels A–I correspond to immunofluorescence detection in fixed cells. PrP (green) was stained using 3F4 mAb, nuclei (blue) were stained with Hoescht and Golgi (red) with anti-βCOP antibody. Panels J–R display the fluorescence of the corresponding YFP fusion PrPs in living cells. Mutants M134S, M154S, and M213L were used as negative controls; G123P and A117V as negative and positive controls for CtmPrP, and C214A as control for forms lacking intramolecular disulfide bond.
      To test whether this intracellular PrP distribution was associated with ER stress, we tested the levels of key components of the unfolded protein response pathway following the expression of HaPrP mutants. Expression of WT HaPrP and of its control M134S, M154S, and M213L mutants did not affect the levels of ER chaperone BiP/Grp78 and of the apoptosis inducer transcription factor CHOP (GADD153) (Fig. 4). However, expression of all PrPα3M mutants along with the A117V construct resulted in a significant increase of BiP/Grp78 and CHOP levels (Fig. 4). We also analyzed the levels of PDI, an ER chaperone that catalyzes thiol-disulfide exchanges and assists in disulfide bridge formation during the folding of secretory proteins. We found that the levels of PDI did not differ significantly between WT HaPrP and all of the mutants tested (Fig. 4). Taken together these results show the selective activation of the ER stress response upon accumulation of CtmPrP conformers.
      Figure thumbnail gr4
      FIGURE 4PrPα3M elicit an ER stress response. A, immunoblot analysis of Grp78, CHOP, and PDI ER stress markers associated with the expression of HaPrP mutants. β-Actin signal was used as control for quantification. B, relative quantification of Grp78, CHOP, and PDI levels in HpL cells after 48 h of transfection with WT HaPrP and its M134S, M154S, M206S, M213S, DS, M213L, A117V, and C214A mutants. Mutants M134S, M154S, and M213L were used as negative controls; A117V as control for CtmPrP and C214A as control for forms lacking intramolecular disulfide bond.

      PrPα3M Mutants Lack the Conventional Intramolecular Disulfide Bond

      A similar toxicity as observed here for the PrPα3M mutants has been described previously for the MoPrPC213A mutant and ascribed to a failure of the oxidative folding (
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Riemer J.
      • Bulleid N.
      • Herrmann J.M.
      Disulfide formation in the ER and mitochondria: two solutions to a common process.
      ). Indeed, this mutant lacks the intramolecular disulfide bond which is a key determinant for the stability of the α2–α3 subdomain both in vivo and in vitro (
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Yanai A.
      • Meiner Z.
      • Gahali I.
      • Gabizon R.
      • Taraboulos A.
      Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.
      ,
      • Adrover M.
      • Pauwels K.
      • Prigent S.
      • de Chiara C.
      • Xu Z.
      • Chapuis C.
      • Pastore A.
      • Rezaei H.
      Prion fibrillization is mediated by a native structural element that comprises helices H2 and H3.
      ,
      • Lee S.
      • Eisenberg D.
      Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process.
      ).
      To understand better the similarities between the mutant lacking the disulfide bond and the PrPα3M mutants here, we generated the HaPrPC214A mutant, analyzed its toxicity, and tested whether it accumulates as CtmPrP. Fig. 2A shows that expression HaPrPC214A is as cytotoxic as that of HaPrPα3M mutants, in agreement with previous observations (
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ). On the other hand, Fig. 2E shows that microsomes of cells expressing HaPrPC214A behaved in the protein protection assay as microsomes from PrPα3M and A117V mutants. These data support that the high toxicity of all these mutants is related to the adoption of CtmPrP topologies.
      Then, we analyzed the state of the intramolecular disulfide bond for the PrPα3M mutants using the differential reactivity of free and oxidized thiol groups with Oregon Green-iodoacetamide (Fig. 5A). Lysates of transfected cells which we first denatured in the absence and presence of DTT were then treated with Oregon Green-iodoacetamide for the irreversible labeling of free thiol groups. Next, PrP was immunoprecipitated with mAb 3F4 and immunoblotted with anti-Oregon Green/fluorescein antibody to detect only the fraction with free thiols. This assay clearly showed that WT HaPrP and its M134S, M154S, and M213L mutants formed stable disulfide bonds and did not interact with Oregon Green-iodoacetamide in the absence of DTT (Fig. 5). In contrast, the PrPα3M, A117V, and C214A mutants were labeled with Oregon Green-iodoacetamide in the absence of DTT (Fig. 5B). Thus, these data suggest that a key structural feature of toxic mutants forming CtmPrP could be the presence of free thiols. To confirm this observation, we studied the effect of the DS mutation on HaPrPG123P, a mutant with described impaired capacity to generate transmembrane forms (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Ashok A.
      • Hegde R.S.
      Selective processing and metabolism of disease-causing mutant prion proteins.
      ). HaPrPG123PDS shared with HaPrPDS the expression as a single band, but behaved as HaPrPG123P in topology assay (Figs. 2, F and G, 3, and 4). Importantly, HaPrPG123PDS expression produced cell viability values similar to those of WT HaPrP and HaPrPG123P. Taken together, these data support a direct relation between the lack of intramolecular disulfide bond and the formation of toxic CtmPrP.
      Figure thumbnail gr5
      FIGURE 5Detection of free thiols in HaPrPα3M and other mutants forming CtmPrP. Oregon Green-iodoacetamide labeling of PrP in cell lysates pretreated in the (A) presence and (B) absence of DTT shown by mAb 3F4 capture and anti-Oregon Green development. DTT pretreatment permits labeling of all PrP chains (both disulfide-bonded and -free species), whereas labeling in the absence of DTT selects PrP chains containing thiol-free groups. Mutants M134S, M154S, and M213L were used as negative controls; A117V as control for CtmPrP and C214A as control for forms lacking intramolecular disulfide bond.

      DISCUSSION

      The efficient production of secreted PrPC starts with the co-translational translocation to the ER followed by a series of concerted processing including the cleavage of the N-terminal signal sequence, the addition of glycan chains at two facultative sites, the formation of an intramolecular disulfide bond, and a transamidation at the C terminus to add the GPI moiety (
      • Aguzzi A.
      • Calella A.M.
      Prions: protein aggregation and infectious diseases.
      ). Of these post-translational modifications, glycosylation and disulfide bond formation depend on the cellular redox state (
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Orsi A.
      • Sitia R.
      Interplays between covalent modifications in the endoplasmic reticulum increase conformational diversity in nascent prion protein.
      ). Impairing the ER oxidative environment or mutating the cysteines yields intracellular, diglycosylated PrP chains lacking the disulfide bond, which resembles the PrPα3M mutants described here (
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Orsi A.
      • Sitia R.
      Interplays between covalent modifications in the endoplasmic reticulum increase conformational diversity in nascent prion protein.
      ,
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Yanai A.
      • Meiner Z.
      • Gahali I.
      • Gabizon R.
      • Taraboulos A.
      Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.
      ). In this work we demonstrated the interplay between oxidative folding and the formation of the toxic CtmPrP. Introduction of polar substitutions at the α3 methionines, singly or combined, precluded the formation of the intramolecular disulfide bond and dictated the stabilization of highly toxic CtmPrP topologies that killed cells. Importantly, the absence of the disulfide bond in CtmPrP and its escape from the quality control checkpoints support the impairment of the oxidative folding as key pathogenic event for the of toxic PrP forms during normal aging.
      CtmPrP has been traditionally viewed as a transmembrane form whose translocation is governed by the hydrophobic region, resulting in a cytosolic N-terminal domain containing the signal sequence (
      • Hegde R.S.
      • Mastrianni J.A.
      • Scott M.R.
      • DeFea K.A.
      • Tremblay P.
      • Torchia M.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      A transmembrane form of the prion protein in neurodegenerative disease.
      ,
      • Hegde R.S.
      • Tremblay P.
      • Groth D.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      Transmissible and genetic prion diseases share a common pathway of neurodegeneration.
      ,
      • Harris D.A.
      Trafficking, turnover and membrane topology of PrP.
      ). However, recent experiments in cell cultures showed that CtmPrP lacked the N-terminal signal sequence, supporting a model of transbilayer post-translocation slippage as observed with recombinant PrP lacking disulfide bond and lipid vesicles (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ,
      • Shin J.I.
      • Shin J.Y.
      • Kim J.S.
      • Yang Y.S.
      • Shin Y.K.
      • Kweon D.H.
      Deep membrane insertion of prion protein upon reduction of disulfide bond.
      ,
      • Shin J.Y.
      • Shin J.I.
      • Kim J.S.
      • Yang Y.S.
      • Shin Y.K.
      • Kim K.K.
      • Lee S.
      • Kweon D.H.
      Disulfide bond as a structural determinant of prion protein membrane insertion.
      ). Conditions favoring the slippage by trapping the hydrophobic region at the translocon as in A117V may either kinetically delay or impose a distance constraint impeding the formation of the disulfide bond. Also, mutations that may hinder oxidoreductase recognition such as α3M and Y216A may favor the insertion of the N-terminal domain as shown with recombinant chains lacking a disulfide bond (
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Shin J.I.
      • Shin J.Y.
      • Kim J.S.
      • Yang Y.S.
      • Shin Y.K.
      • Kweon D.H.
      Deep membrane insertion of prion protein upon reduction of disulfide bond.
      ,
      • Shin J.Y.
      • Shin J.I.
      • Kim J.S.
      • Yang Y.S.
      • Shin Y.K.
      • Kim K.K.
      • Lee S.
      • Kweon D.H.
      Disulfide bond as a structural determinant of prion protein membrane insertion.
      ).
      Our results also suggest that conditions preventing PrP oxidative folding may favor the formation of CtmPrP and, consequently, promote its deleterious effects. Indeed, both maintenance of the levels and activity of ER oxidoreductases such as Grp58 and PdIa protect against the toxicity of misfolded PrP forms (
      • Hetz C.
      • Russelakis-Carneiro M.
      • Wälchli S.
      • Carboni S.
      • Vial-Knecht E.
      • Maundrell K.
      • Castilla J.
      • Soto C.
      The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity.
      ,
      • Watts J.C.
      • Huo H.
      • Bai Y.
      • Ehsani S.
      • Jeon A.H.
      • Won A.H.
      • Shi T.
      • Daude N.
      • Lau A.
      • Young R.
      • Xu L.
      • Carlson G.A.
      • Williams D.
      • Westaway D.
      • Schmitt-Ulms G.
      Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.
      ). On aging, both levels and activity of ER chaperones cause a decline of oxidative folding (
      • Nuss J.E.
      • Choksi K.B.
      • DeFord J.H.
      • Papaconstantinou J.
      Decreased enzyme activities of chaperones PDI and BiP in aged mouse livers.
      ). Because PrP3αM mutants were tailored to mimic the effects of Met sulfoxidation, conditions favoring such modification on nascent chains would also facilitate the formation of CtmPrP. In this line, ER stress exhaustion concurs with an overproduction of reactive oxygen species which are the major oxidants of Met residues, as those exposed in nascent unfolded chains (
      • Gregersen N.
      • Bross P.
      Protein misfolding and cellular stress: an overview.
      ). Taken together, both decreased efficiency of the oxidative folding and increased probability of sulfoxidation of Met may then explain the aging-associated accumulation of CtmPrP forms in sporadic and inherited diseases.
      The role of disulfide bonds in PrP biology has mainly focused on their contribution to the generation of prions. The analysis of highly pure preparations of PrP27–30, the protease-resistant core of PrPSc, indicated that all Cys were forming disulfide bonds (
      • Hegde R.S.
      • Mastrianni J.A.
      • Scott M.R.
      • DeFea K.A.
      • Tremblay P.
      • Torchia M.
      • DeArmond S.J.
      • Prusiner S.B.
      • Lingappa V.R.
      A transmembrane form of the prion protein in neurodegenerative disease.
      ,
      • Turk E.
      • Teplow D.B.
      • Hood L.E.
      • Prusiner S.B.
      Purification and properties of the cellular and scrapie hamster prion proteins.
      ). But those samples lack CtmPrP forms due to the proteinase K digestion step used in the purification. The first evidence indirectly suggesting a pathogenic role for free thiols was provided by deletion mutants (
      • Muramoto T.
      • DeArmond S.J.
      • Scott M.
      • Telling G.C.
      • Cohen F.E.
      • Prusiner S.B.
      Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an α-helix.
      ). Mice expressing PrPΔ177-200 and PrPΔ201–217 that are unable to forms intramolecular disulfide bonds developed signs and lesions characteristic of neuronal storage disorders. In these mice, truncated PrP was detergent-insoluble in detergent, PK-sensitive, and with migration properties resembling those of PrPα3M mutants. Also, Cys mutants used in cellular studies resulted in PrP forms sharing key properties with PrPα3M mutants, but their topology and toxicity were not addressed (
      • Capellari S.
      • Zaidi S.I.
      • Urig C.B.
      • Perry G.
      • Smith M.A.
      • Petersen R.B.
      Prion protein glycosylation is sensitive to redox change.
      ,
      • Orsi A.
      • Sitia R.
      Interplays between covalent modifications in the endoplasmic reticulum increase conformational diversity in nascent prion protein.
      ,
      • Tabrett C.A.
      • Harrison C.F.
      • Schmidt B.
      • Bellingham S.A.
      • Hardy T.
      • Sanejouand Y.H.
      • Hill A.F.
      • Hogg P.J.
      Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.
      ,
      • Yanai A.
      • Meiner Z.
      • Gahali I.
      • Gabizon R.
      • Taraboulos A.
      Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.
      ).
      The finding that free thiols lead to the formation of CtmPrP has several implications. From the structural point of view, the C-terminal domain has to expand its known conformational repertoire to accommodate the absence of the α2–α3 constraint and a double tether to the membrane (
      • Adrover M.
      • Pauwels K.
      • Prigent S.
      • de Chiara C.
      • Xu Z.
      • Chapuis C.
      • Pastore A.
      • Rezaei H.
      Prion fibrillization is mediated by a native structural element that comprises helices H2 and H3.
      ,
      • Lee S.
      • Eisenberg D.
      Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process.
      ,
      • Maiti N.R.
      • Surewicz W.K.
      The role of disulfide bridge in the folding and stability of the recombinant human prion protein.
      ). Also, the fibrillation of CtmPrP may be impeded by the diglycosylation (
      • Lee S.
      • Eisenberg D.
      Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process.
      ,
      • Bosques C.J.
      • Imperiali B.
      The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment.
      ). But the detergent insolubility of CtmPrP suggests that it may populate distinct aggregate states, adding more structural complexity. Functionally, the aggregation of PrPα3M mutants suggests that CtmPrP may exert its toxic function through an oligomeric thiol trap. Such traps have been described in the regulation of IgM and adiponectin secretions (
      • Anelli T.
      • Alessio M.
      • Bachi A.
      • Bergamelli L.
      • Bertoli G.
      • Camerini S.
      • Mezghrani A.
      • Ruffato E.
      • Simmen T.
      • Sitia R.
      Thiol-mediated protein retention in the endoplasmic reticulum: the role of ERp44.
      ,
      • Anelli T.
      • Ceppi S.
      • Bergamelli L.
      • Cortini M.
      • Masciarelli S.
      • Valetti C.
      • Sitia R.
      Sequential steps and checkpoints in the early exocytic compartment during secretory IgM biogenesis.
      ,
      • Fra A.M.
      • Fagioli C.
      • Finazzi D.
      • Sitia R.
      • Alberini C.M.
      Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.
      ,
      • Guenzi S.
      • Fra A.M.
      • Sparvoli A.
      • Bet P.
      • Rocco M.
      • Sitia R.
      The efficiency of cysteine-mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells.
      ,
      • Reddy M.M.
      • Bell C.L.
      Distinct cellular mechanisms of cholinergic and β-adrenergic sweat secretion.
      ,
      • Sitia R.
      • Neuberger M.
      • Alberini C.
      • Bet P.
      • Fra A.
      • Valetti C.
      • Williams G.
      • Milstein C.
      Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine.
      ,
      • Wang Y.
      • Lam K.S.
      • Yau M.H.
      • Xu A.
      Post-translational modifications of adiponectin: mechanisms and functional implications.
      ). Importantly, given the validation of the PrP chains by the ER quality control systems the assembly of such oligomeric traps must take place upon delivery to a different environment (
      • Ashok A.
      • Hegde R.S.
      Selective processing and metabolism of disease-causing mutant prion proteins.
      ). Whether other components participate in their assembly and/or stabilization and whether these factors display different affinities for the WT or mutant chains remains to be elucidated (
      • Emerman A.B.
      • Zhang Z.R.
      • Chakrabarti O.
      • Hegde R.S.
      Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
      ). Additionally, this unknown lipid-bound conformation of thiol-free PrP could indeed function as the seed for the conversion of PrPC into PrPSc, even in traces amounts (
      • Lucassen R.
      • Nishina K.
      • Supattapone S.
      In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups.
      ,
      • Wang F.
      • Wang X.
      • Yuan C.G.
      • Ma J.
      Generating a prion with bacterially expressed recombinant prion protein.
      ). This could provide a mechanistic explanation for the spontaneous generation of pathogenic forms of PrP in sporadic human diseases.

      Acknowledgments

      We thank Rosa Sánchez for technical assistance and Silvia Zorrilla for advice.

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