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DNA Converts Cellular Prion Protein into the β-Sheet Conformation and Inhibits Prion Peptide Aggregation*

Open AccessPublished:December 28, 2001DOI:https://doi.org/10.1074/jbc.M106707200
      The main hypothesis for prion diseases proposes that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which in most cases undergoes aggregation. In an organism infected with PrPSc, PrPC is converted into the β-sheet form, generating more PrPSc. We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23–231)) converts it from an α-helical conformation (cellular isoform) into a soluble, β-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.
      PrPSc
      prion protein from scrapie
      PrPC
      cellular prion protein
      mPrP
      murine prion protein
      bis-ANS
      4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid
      MES
      4-morpholineethanesulfonic acid
      LS
      light scattering
      Transmissible spongiform encephalopathies are fatal degenerative diseases of the central nervous system characterized by loss of motor control, dementia, and paralysis (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Weissman C.
      ,
      • Caughey B.
      ). They include scrapie of sheep and goats, bovine spongiform encephalopathy in cattle, and several human diseases such as Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Weissman C.
      ,
      • Caughey B.
      ,
      • Will R.G.
      • Ironside J.W.
      • Zeidler M.
      • Cousens S.N.
      • Estibeiro K.
      • Alperovitch A.
      • Poser S.
      • Pocchiari M.
      • Hofman A.
      • Smith P.G.
      ,
      • Prusiner S.B.
      ,
      • Bons N.
      • Mestre-Frances N.
      • Belli P.
      • Cathala F.
      • Gajdusek D.C.
      • Brown P.
      ). Increasing evidence suggests that a new variant of Creutzfeldt-Jakob disease is due to consumption of bovine spongiform encephalopathy-contaminated food (
      • Will R.G.
      • Ironside J.W.
      • Zeidler M.
      • Cousens S.N.
      • Estibeiro K.
      • Alperovitch A.
      • Poser S.
      • Pocchiari M.
      • Hofman A.
      • Smith P.G.
      ). The “protein-only” hypothesis formulated by Prusiner and co-workers (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Prusiner S.B.
      ) considers that the infectious form of the prion (PrPSc,1 from scrapie) has an amino acid sequence identical to a normal host protein (PrPC). Infection of an organism with PrPScwould result in the conversion of PrPC into a conformational image of itself resulting in PrPScaggregation in the brain (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Weissman C.
      ,
      • Prusiner S.B.
      ). The unique characteristic of the infectious prion agent is the lack of a coding nucleic acid (
      • Alper T.
      • Cramp W.A.
      • Haig D.A.
      • Clarke M.C.
      ). It has been proposed that a predominantly α-helical structure in the PrPC converts into PrPSc protein that is rich in β-sheet, especially in region 90–145 (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Pan K.H.
      • Baldwin M.
      • Nguyen J.
      • Gasset M.
      • Serban A.
      • Groth D.
      • Mehlhorn I.
      • Huang Z.
      • Fletterick J.
      • Cohen F.E.
      ,
      • Huang Z.
      • Prusiner S.B.
      • Cohen F.E.
      ,
      • Jackson G.S.
      • Hosszu L.L.
      • Power A.
      • Hill A.F.
      • Kenney J.
      • Saibil H.
      • Craven C.J.
      • Waltho J.P.
      • Clarke A.R.
      • Collinge J.
      ). Two recent reports (
      • Supattapone S.
      • Bosque P.
      • Muramoto T.
      • Wille H.
      • Aargard C.
      • Peretz D.
      • Nguyen H.
      • Heinrich C.
      • Torchia M.
      • Safar J.
      • Cohen F.E.
      • DeArmond S.J.
      • Prusiner S.B.
      • Scott M.
      ,
      • Kaneko K.
      • Ball H.L.
      • Wille H.
      • Zhang H.
      • Groth D.
      • Torchia M.
      • Tremblay P.
      • Safar J.
      • Prusiner S.B.
      • DeArmond S.J.
      • Prusiner S.B.
      • DeArmond S.J.
      • Baldwin M.A.
      • Cohen F.E.
      ) corroborate the hypothesis that most of the conformational change involving the appearance of the β-structure occurs in the segment 90–145. A redacted version of PrP lacking residues 23–88 and 141–176 can cause prion disease (
      • Supattapone S.
      • Bosque P.
      • Muramoto T.
      • Wille H.
      • Aargard C.
      • Peretz D.
      • Nguyen H.
      • Heinrich C.
      • Torchia M.
      • Safar J.
      • Cohen F.E.
      • DeArmond S.J.
      • Prusiner S.B.
      • Scott M.
      ), and the 55-residue peptide (MoPrP-(89–143)) carrying the P101L substitution was able to initiate Gerstmann-Sträussler-Scheinker syndrome disease in transgenic mice (
      • Kaneko K.
      • Ball H.L.
      • Wille H.
      • Zhang H.
      • Groth D.
      • Torchia M.
      • Tremblay P.
      • Safar J.
      • Prusiner S.B.
      • DeArmond S.J.
      • Prusiner S.B.
      • DeArmond S.J.
      • Baldwin M.A.
      • Cohen F.E.
      ). The importance of this region in the pathogenesis was first recognized when it was discovered that the peptide 106–126 is highly neurotoxic (
      • Forloni G.
      • Angeretti N.
      • Chiesa R.
      • Monzani E.
      • Salmona M.
      • Bugiani O.
      • Tagliavini F.
      ).
      The protein-only theory gained great support with the finding that PrP knockout mice are resistant to infection with prions (
      • Büeler H.
      • Fisher M.
      • Lang Y.
      • Bluethmann H.
      • Lipp H.P.
      • DeArmond S.J.
      • Prusiner S.B.
      • Auet M.
      • Weissmann C.
      ). More recently, yeast phenotypes were demonstrated to have prion properties (,
      • Wickner R.B.
      • Edskes H.K.
      • Makkelein M.L.
      • Taylor K.L.
      • Moriyama H.
      ,
      • Serio T.R.
      • Cashikar A.G.
      • Kowal A.S.
      • Sawicki G.J.
      • Moslehi J.J.
      • Serpell L.
      • Arnsdorf M.F.
      • Lindquist S.L.
      ,
      • Chien P.
      • Weissman J.S.
      ). The mechanism of prion conversion remains unknown, but most models predict an autocatalytic process (
      • Cohen F.E.
      • Prusiner S.B.
      ,
      • Weissman C.
      ,
      • Prusiner S.B.
      ,
      • Jarrett J.T.
      • Lansbury Jr., P.T.
      ,
      • Come J.
      • Fraser P.E.
      • Lansbury Jr., P.T.
      ). The availability of NMR solution structures of different constructs of the cellular form of PrP has opened the possibility to address questions related to the conformation and dynamics of the cellular protein and its interaction with environmental factors (
      • Riek R.
      • Hornemann S.
      • Wider G.
      • Billeter M.
      • Glockshuber R.
      • Wütrich K.
      ,
      • Donne D.G.
      • Viles J.H.
      • Groth D.
      • Mehlhorn I.
      • James T.L.
      • Cohen F.E.
      • Prusiner S.B.
      • Wright P.E.
      • Dyson H.J.
      ,
      • Zahn R.
      • Liu A.
      • Luhrs T.
      • Riek R.
      • von Schroetter C.
      • Lopez Garcia F.
      • Billeter M.
      • Calzolai L.
      • Wider G.
      • Wuthrich K.
      ,
      • Viles J.H.
      • Donne D.
      • Kroon G.
      • Prusiner S.B.
      • Cohen F.E.
      • Dyson H.J.
      • Wright P.E.
      ,
      • Kelly J.W.
      ). However, many questions remain unanswered. 1) Why the low efficiency of infection (ratio 1:100,000)? 2) Can recombinant PrPC molecules cause disease in the absence of infectious prion? 3) Are there adjuvant factors? In fact, concerning the last question, recent studies (
      • Prusiner S.B.
      ,
      • Telling G.C.
      • Scott M.
      • Mastrianni J.
      • Gabizon R.
      • Torchia M.
      • Cohen F.E.
      • DeArmond S.J.
      • Prusiner S.B.
      ) indicate that another macromolecule (provisionally designated protein X) may interact with the prion protein acting as an adjuvant factor.
      We find that some nucleic acid sequences bind with high affinity to the mPrP-(23–231) converting it to the β-sheet isoform. DNA also binds to prion sequences containing the region that undergoes the transition to β-sheet, modulating their aggregation. Previous studies have shown that PrP may interact with sulfated glycans (
      • Caughey B.
      • Brown K.
      • Raymond G.J.
      • Katzenstein G.E.
      • Thresher W.
      ), DNA (
      • Nandi P.K.
      • Leclerc E.
      ), and RNA (
      • Gabus C.
      • Derrington E.
      • Leblanc P.
      • Chnaiderman J.
      • Dormont D.
      • Swietnicki W.
      • Morillas M.
      • Surewicz W.K.
      • Marc D.
      • Nandi P.
      • Darlix J.L.
      ). We also find that there is a dependence on the DNA sequence for binding to the murine prion protein. At high DNA-to-protein ratios, aggregation is inhibited. When the aggregating sequence (S) is present in excess of DNA, the aggregates rescue the protein from the DNA. Based on these findings, we propose a mechanism for the participation of an adjuvant factor (factor X), likely a nucleic acid.

      Acknowledgments

      We are grateful to Peter H. von Hippel, Clifford R. Robinson, and Martha Sorenson for critical comments and advice and to Ralph Zahn for the pRSETA construct containing mPrP-(23–231) cDNA.

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