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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.
      ,
      • 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.
      ).
      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.

      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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