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Reactive Oxygen Species-mediated β-Cleavage of the Prion Protein in the Cellular Response to Oxidative Stress*

  • Nicole T. Watt
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • David R. Taylor
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • Andrew Gillott
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • Daniel A. Thomas
    Footnotes
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • W. Sumudhu S. Perera
    Footnotes
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • Nigel M. Hooper
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-113-343-3163; Fax: 44-113-343-3167;
    Affiliations
    Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
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  • Author Footnotes
    * This work was supported by the Medical Research Council of Great Britain, European Union Grant QLG3-CT-2001-02353, and the Wellcome Trust (Bioimaging Facility, University of Leeds). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    1 Recipient of a Biotechnology and Biological Sciences Research Council studentship.
    2 Recipient of an Emma and Leslie Reid studentship from the University of Leeds. Present address: Institut für Zoologie, Abteilung Molekulare Zellbiologie, Universität Mainz, D-55128 Mainz, Germany.
      The cellular prion protein (PrPC) is critical for the development of prion diseases. However, the physiological role of PrPC is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrPC is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed β-cleavage. Here we show that ROS-mediated β-cleavage of cell surface PrPC occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPΔoct, failed to undergo ROS-mediated β-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPΔoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated β-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the β-cleavage of PrPC is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.
      Prion diseases or transmissible spongiform encephalopathies are a group of neurodegenerative disorders including scrapie in sheep, bovine spongiform encephalopathy in cattle, Creutzfeldt-Jakob disease, and Gerstmann-Sträussler-Scheinker disease in humans (
      • Prusiner S.B.
      ). In prion diseases, the normal cellular form of the prion protein (PrPC)
      The abbreviations used are:
      PrPC
      cellular form of the prion protein
      PrPSc
      infectious, protease resistant form of PrP
      ALLM
      N-acetyl-Leu-Leu-Met-aldehyde
      FCS
      fetal calf serum
      PBS
      phosphate-buffered saline
      PNGase F
      Peptide:N-glycosidase F
      ROS
      reactive oxygen species
      wtPrP
      wild type PrP
      GPI
      glycosylphosphatidylinositol
      4The abbreviations used are:PrPC
      cellular form of the prion protein
      PrPSc
      infectious, protease resistant form of PrP
      ALLM
      N-acetyl-Leu-Leu-Met-aldehyde
      FCS
      fetal calf serum
      PBS
      phosphate-buffered saline
      PNGase F
      Peptide:N-glycosidase F
      ROS
      reactive oxygen species
      wtPrP
      wild type PrP
      GPI
      glycosylphosphatidylinositol
      undergoes a conformational conversion to the β-sheet-rich scrapie isoform (PrPSc) that is partially resistant to protease digestion. Although PrPC is critical for the development of prion disease through its conversion into PrPSc (
      • Bueler H.
      • Aguzzi A.
      • Sailer A.
      • Greiner R.A.
      • Autenried P.
      • Aguet M.
      • Weissmann C.
      ,
      • Prusiner S.B.
      • Groth D.
      • Serban A.
      • Koehler R.
      • Foster D.
      • Torchia M.
      • Burton D.
      • Yang S.L.
      • DeArmond S.J.
      ), the physiological role of PrPC is less clear, and thus it is uncertain whether prion diseases are, in part, due to the loss of a normal neuro-protective function of PrPC (
      • Hetz C.
      • Maundrell K.
      • Soto C.
      ). In the brains of animals at the terminal stage of illness, there is a marked decrease of PrPC, supporting the hypothesis that loss of function of PrPC may play a role in the pathogenesis of prion diseases (
      • Yokoyama T.
      • Kimura K.M.
      • Ushiki Y.
      • Yamada S.
      • Morooka A.
      • Nakashiba T.
      • Sassa T.
      • Itohara S.
      ).
      Among the neuroprotective functions of PrPC are roles in copper homeostasis and the cellular resistance to oxidative stress (
      • Vassallo N.
      • Herms J.
      ,
      • Roucou X.
      • Gains M.
      • LeBlanc A.C.
      ). PrPC binds Cu2+ ions, primarily within the N-terminal octapeptide repeats (
      • Brown D.R.
      • Qin K.
      • Herms J.W.
      • Madlung A.
      • Manson J.
      • Strome R.
      • Fraser P.E.
      • Kruck T.
      • von Bohlen A.
      • Schulz-Schaeffer W.
      • Giese A.
      • Westaway D.
      • Kretzschmar H.
      ,
      • Miura T.
      • Hori-i A.
      • Mototani H.
      • Takeuchi H.
      ,
      • Viles J.H.
      • Cohen F.E.
      • Prusiner S.B.
      • Goodin D.B.
      • Wright P.E.
      • Dyson H.J.
      ), undergoes endocytosis upon exposure of cells to Cu2+ (
      • Pauly P.C.
      • Harris D.A.
      ,
      • Perera W.S.S.
      • Hooper N.M.
      ), and modulates neuronal Cu2+ content (
      • Brown D.R.
      ), implicating PrPC in cellular copper metabolism. Cells deficient in PrPC are less viable in culture compared with cells expressing wild-type PrP (wtPrP) and are more susceptible to oxidative damage and toxicity caused by reactive oxygen species (ROS) (
      • Brown D.R.
      • Schulz-Schaeffer W.J.
      • Schmidt B.
      • Kretzschmar H.A.
      ,
      • Kuwahara C.
      • Takeuchi A.M.
      • Nishimura T.
      • Haraguchi K.
      • Kubosaki A.
      • Matsumoto Y.
      • Saeki K.
      • Yokoyama T.
      • Itohara S.
      • Onodera T.
      ,
      • White A.R.
      • Collins S.J.
      • Maher F.
      • Jobling M.F.
      • Stewart L.R.
      • Thyer J.M.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ,
      • Zeng F.
      • Watt N.T.
      • Walmsley A.R.
      • Hooper N.M.
      ), implicating PrPC in the cellular response to oxidative stress. However, the mechanism by which PrPC mediates this protective effect is not known.
      PrPC is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that undergoes a variety of proteolytic processing events. The protein can be cleaved at amino acids 110 and 111 to produce a 17-kDa C-terminal fragment C1 and a corresponding N-terminal fragment N1 (
      • Pan K.-M.
      • Stahl N.
      • Prusiner S.B.
      ,
      • Shyng S.-L.
      • Huber M.T.
      • Harris D.A.
      ,
      • Chen S.G.
      • Teplow D.B.
      • Parchi P.
      • Teller J.K.
      • Gambetti P.
      • Autilio-Gambetti L.
      ,
      • Jimenez-Huete A.
      • Lievens P.M.J.
      • Vidal R.
      • Piccardo P.
      • Ghetti B.
      • Tagliavini F.
      • Frangione B.
      • Prelli F.
      ). This processing has been referred to as α-cleavage (
      • Mange A.
      • Beranger F.
      • Peoc'h K.
      • Onodera T.
      • Frobert Y.
      • Lehmann S.
      ) and may be mediated by ADAM 10 and ADAM 17, members of the ADAM (adisintegrin and metalloprotease) family (
      • Vincent B.
      • Paitel E.
      • Saftig P.
      • Frobert Y.
      • Hartmann D.
      • De Strooper B.
      • Grassi J.
      • Lopez-Perez E.
      • Checler F.
      ). PrPC can also be cleaved within or adjacent to the octapeptide repeats to generate a 21-kDa C-terminal fragment C2 and the corresponding N-terminal fragment N2 (
      • Pan K.-M.
      • Stahl N.
      • Prusiner S.B.
      ,
      • Jimenez-Huete A.
      • Lievens P.M.J.
      • Vidal R.
      • Piccardo P.
      • Ghetti B.
      • Tagliavini F.
      • Frangione B.
      • Prelli F.
      ,
      • Taraboulos A.
      • Raeber A.J.
      • Borchelt D.R.
      • Serban D.
      • Prusiner S.B.
      ). This cleavage event appears to be mediated by ROS (
      • McMahon H.E.
      • Mange A.
      • Nishida N.
      • Creminon C.
      • Casanova D.
      • Lehmann S.
      ) and has been termed β-cleavage (
      • Mange A.
      • Beranger F.
      • Peoc'h K.
      • Onodera T.
      • Frobert Y.
      • Lehmann S.
      ). In addition, we have recently shown that PrPC is proteolytically shed from the cell surface by a zinc metalloprotease that has similar properties to the α-secretase cleavage of the Alzheimer amyloid precursor protein (
      • Parkin E.T.
      • Watt N.T.
      • Turner A.J.
      • Hooper N.M.
      ). Understanding the role of these proteolytic cleavages and of the fragments generated is critical to a full understanding of the biological functions of PrP and may also impact on the role of the protein in prion diseases.
      However, the role of the β-cleavage in the function of PrPC has not been addressed. In the current study, we show that PrP expressed in the human neuroblastoma SH-SY5Y cell line undergoes both α- and β-cleavage and that β-cleavage is increased upon exposure of the cells to ROS, occurs at the cell surface, and can be inhibited by a free radical quencher. We also show that β-cleavage does not occur in a mutant of PrP that lacks the octapeptide repeats (PrPΔoct) nor in two disease-associated mutants of PrP (PG14 and A116V). This lack of β-cleavage of the PrP mutants correlates with the sensitivity of cells to ROS, indicating that this cleavage event may be part of the mechanism by which PrPC protects cells against oxidative stress.

      EXPERIMENTAL PROCEDURES

      cDNA Constructs and Cell Culture—The construction of wtPrP, PrPΔoct, and PG14 in pIRESneo has been described previously (
      • Perera W.S.S.
      • Hooper N.M.
      ). A116V was generated from wtPrP using the QuikChange site-directed mutagenesis kit (Stratagene) with the following primers: sense, 5′-CAGGGGCTGCGGTAGCTGGGGGCAGTAG-3′; antisense, 5′-CTACTGCCCCAGCTACCGCAGCCCCTG-3′. The resulting construct was verified by DNA sequencing. Human neuroblastoma SH-SY5Y cells were cultured and transfected by electroporation, and pooled, stable cell lines were obtained by antibiotic selection as described previously (
      • Walmsley A.R.
      • Zeng F.
      • Hooper N.M.
      ). Copper was routinely administered to the cells as CuSO4 in the presence of fetal calf serum (FCS) to provide a source of both albumin and histidine for the Cu2+ to complex to. When the cells had reached confluence, the monolayer was washed twice with Opti-MEM before incubation with the relevant compounds for the specified periods of exposure in Opti-MEM. Cells were harvested into phosphate-buffered saline (PBS; 1.5 mm KH2PO4, 2.7 mm Na2HPO4, 150 mm NaCl, pH 7.4) pelleted by centrifugation at 1000 × g for 5 min, and resuspended in lysis buffer (10 mm Tris/HCl, pH 7.8, 0.5% (w/v) sodium deoxycholate, 0.5% (v/v) Nonidet P-40, 100 mm NaCl, 10 mm EDTA, supplemented with complete protease inhibitor mixture). The protein content of each lysate was determined using bicinchoninic acid in a microtiter plate assay with bovine serum albumin as a standard (
      • Smith P.K.
      • Krohn R.I.
      • Hermanson G.T.
      • Mallia A.K.
      • Gartner F.H.
      • Provenzano M.D.
      • Fujimoto E.K.
      • Goeke B.J.
      • Olson B.J.
      • Klenk D.C.
      ).
      Surface Biotinylation and Immunoprecipitation—Cells at confluence were incubated for1hat4°C with 0.5 mg/ml Biotin sulfo-N-hydroxy-succinimide (NHS), washed three times with 50 mm glycine to quench the biotinylation reaction, and then incubated for various times at 37 °C in the absence or presence of 10 μm CuSO4 and 100 μm H2O2 in Opti-MEM. Cell lysates were made 1% (w/v) with respect to N-lauroylsarcosine and incubated for 30 min with 0.5% (w/v) protein A-Sepharose. The protein A-Sepharose was pelleted by centrifugation for 1 min at 13,000 × g, and the supernatant was incubated overnight at 4 °C with 0.1% (v/v) 3F4 antibody. Protein A-Sepharose was added to 0.5% (w/v), and incubation continued for 1 h. The immunocomplexes were pelleted by centrifugation at 13,000 × g for 1 min and washed three times with 150 mm NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mm Tris/HCl, pH 8.0, 1% (v/v) Nonidet P-40. The remaining pellet was resuspended in dissociation buffer for analysis by SDS-PAGE and Western blot. To measure copper-induced endocytosis, biotin-labeled cells were incubated for 30 min at 37 °C in the absence or presence of 100 μm CuSO4 presented as a histidine chelate. PrP remaining at the cell surface was removed by digestion with trypsin as previously described (
      • Perera W.S.S.
      • Hooper N.M.
      ).
      Enzymic Deglycosylation, SDS-PAGE, and Western Blot Analysis—Samples were deglycosylated with peptide:N-glycosidase F (PNGase F) (Europa Bioproducts, Ely, UK) for 16 h at 37 °C as described previously (
      • Walmsley A.R.
      • Zeng F.
      • Hooper N.M.
      ). Where indicated, samples were digested with 5 μg/ml proteinase K for1hat37°C. Samples (containing 15 μg of total protein) or immunoprecipitates were resolved by electrophoresis through 14.5% polyacrylamide gels. For Western blot analysis, resolved proteins were transferred to a Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was blocked by incubation for 1 h with PBS containing 0.1% (v/v) Tween 20 and 5% (w/v) dried milk powder. Incubations with primary antibodies 3F4 (Signet Laboratories, Inc., Dedham, MA), SAF32 (Cayman Chemical, Ann Arbor, MI), 6H4 (Prionics, Zurich, Switzerland), or anti-actin and peroxidase-conjugated secondary antibodies were performed for1hinthesame buffer. Incubation with peroxidase-conjugated streptavidin was performed for1hinPBS containing 0.1% (v/v) Tween 20. Bound peroxidase conjugates were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences).
      Recombinant Calpain Activity Assay—The activity of 20 nm recombinant Calpain-2 (Calbiochem) was measured using 5 μm (5-aminomethylfluorescein)-Gly-Gly-Gly-Gln-Leu-Tyr-Gly-Gly(Nβ-(2,4-dinitrophenyl)-l-2,3-diaminopropionic acid)-Arg-Arg-Lys(tetramethylrhodamine)NH2 (a gift from GlaxoSmithKline, Harlow, UK) in 60 mm imidazole/HCl, 5 mml-cysteine, 2.5 mm glutathione (reduced), and 5 mm CaCl2, pH 7.3. The broad spectrum calpain inhibitor N-acetyl-Leu-Leu-Met-aldehyde (ALLM) resuspended in either Me2SO or EtOH was added at 1, 10, or 100 μm. Activity was recorded as fluorescence released following cleavage of the substrate over 1 h using a Synergy HT (Bio-Tek) with excitation at 485 nm and emission at 528 nm. The data wereexpressedasthepercentageinhibitionofactivitycomparedwiththeuninhibited control.
      Immunofluorescence Microscopy—Cells were seeded onto coverslips and grown to 50% confluence. The fate of cell surface PrPC was monitored by prelabeling cells with antibody 3F4 for 30 min at 4 °C. Cells were then incubated in Dulbecco's PBS in the presence or absence of 10 μg/ml Bacillus thuringiensis phosphatidylinositol-specific phospholipase C for 30 min at 37 °C. Cells were then fixed with 4% (v/v) paraformaldehyde, 0.1% (v/v) glutaraldehyde in PBS for 15 min and blocked overnight in PBS containing 3% (v/v) goat serum. Finally, coverslips were incubated with AlexaFluor 488® rabbit anti-mouse IgG (Molecular Probes, Inc., Eugene, OR) for 1 h and mounted on slides using fluoromount G mounting medium (SouthernBiotech). Individual cells were visualized using a DeltaVision Optical Restoration Microscopy System (Applied Precision Inc.). Data were collected from 30–40 0.1-μm-thick optical sections, and three-dimensional data sets were deconvolved using the softWoRx program (Applied Precision Inc.). The images represent individual Z-slices corresponding to the middle of the cell.
      Assessment of Cell Number by Hoescht 33342 Staining—Cells (1 × 104/well) in 96-well tissue culture plates were cultured overnight in serum-free medium. After 24 h, this was replaced with 5% FCS-containing medium supplemented with H2O2 (100 μm), CuSO4 (8 μm), or both reagents. After a further 48 h, the cells were fixed in 70% ethanol at room temperature for 5 min, and the adherent cell monolayers were stained with the DNA-binding fluorochrome Hoescht 33342 (8.8 μm). Once dry, the fluorescence of each well was measured on a Synergy HT (Bio-Tek) (350-nm excitation and 450-nm emission wavelengths) in order to determine the cell number in each well.
      Measurement of Intracellular Oxidative Activity and Glutathione Peroxidase Activity—The level of intracellular free radicals was determined following exposure of the cells to H2O2 (100 μm), CuSO4 (8 μm), or both reagents in 5% FCS-containing medium for 6 h using 100 μm dihydrodichlorofluorescein diacetate as described previously (
      • Zeng F.
      • Watt N.T.
      • Walmsley A.R.
      • Hooper N.M.
      ). Glutathione peroxidase activity was measured using 50 mm Na2HPO4/NaH2PO4, pH 7.0, 1 mm EDTA, 1 mm NaN3, 0.2 mm NADPH, 1 mm glutathione, and 1 unit/ml glutathione reductase at room temperature upon the addition of 0.1 ml of cumene hydroperoxide (1.5 mm) as described previously (
      • Zeng F.
      • Watt N.T.
      • Walmsley A.R.
      • Hooper N.M.
      ).
      Statistical Analysis—All analyses were subject to Kruskal-Wallis nonparametric one-way analysis of variance. p < 0.001 were considered highly significant. Changes in cell number, intracellular radical generation, and glutathione peroxidase activity in the cells expressing the mutant PrPs are all compared against wtPrP-expressing cells.

      RESULTS

      PrPC in SH-SY5Y Cells Is Subject to α- and β-Cleavages—The proteolytic processing of murine PrPC containing the 3F4 epitope (wtPrP) stably expressed in the human neuroblastoma SH-SY5Y cell line was examined using antibodies that recognize different epitopes in the protein (Fig. 1). To remove the problems of interpreting the immunoblots because of the variable glycosylation states of full-length PrP and of the C-terminal fragments, samples were deglycosylated prior to immunoblotting. Antibody SAF32, which recognizes an epitope within the octapeptide repeats, as expected detected full-length PrP but neither the C1 nor C2 fragments in the cell lysate (Fig. 1B). Antibody 3F4, which recognizes the engineered epitope MHKM (residues 108–111 of murine PrP), detected both full-length PrP and the C2 fragment of molecular mass 21 kDa but not the C1 fragment, since α-cleavage destroys the epitope recognized by this antibody (Fig. 1C) (
      • Chen S.G.
      • Teplow D.B.
      • Parchi P.
      • Teller J.K.
      • Gambetti P.
      • Autilio-Gambetti L.
      ,
      • Jimenez-Huete A.
      • Lievens P.M.J.
      • Vidal R.
      • Piccardo P.
      • Ghetti B.
      • Tagliavini F.
      • Frangione B.
      • Prelli F.
      ). Antibody 6H4, which recognizes an epitope in the C-terminal half of the protein (residues 144–152), detected full-length PrP, C2, and the C1 fragment of molecular mass 17 kDa (Fig. 1D). Although the C1 fragment was detected at a similar level of intensity as full-length PrP, the C2 fragment was present at a significantly lower level. These data indicate that in SH-SY5Y cells PrPC is subject to both α- and β-proteolytic cleavages to generate C1 and C2 fragments, respectively, as reported for other cell lines and in brain tissue (
      • Shyng S.-L.
      • Huber M.T.
      • Harris D.A.
      ,
      • Chen S.G.
      • Teplow D.B.
      • Parchi P.
      • Teller J.K.
      • Gambetti P.
      • Autilio-Gambetti L.
      ,
      • Jimenez-Huete A.
      • Lievens P.M.J.
      • Vidal R.
      • Piccardo P.
      • Ghetti B.
      • Tagliavini F.
      • Frangione B.
      • Prelli F.
      ,
      • Mange A.
      • Beranger F.
      • Peoc'h K.
      • Onodera T.
      • Frobert Y.
      • Lehmann S.
      ).
      Figure thumbnail gr1
      FIGURE 1PrPC in SH-SY5Y cells is subject to both α- and β-cleavages. A, schematic diagram of the proteolysis of PrPC and the epitopes recognized by the antibodies used in this study. Mature, full-length PrPC is shown with its C-terminal GPI anchor, two N-linked glycosylation sites (residues 180 and 196, lollipops), and the octapeptide repeat region (shaded). The epitopes for antibody SAF32 (within the octapeptide repeats), 3F4 (residues 108–111), and 6H4 (residues 144–152) are indicated. The two cleavage sites generating N1/C1 (α-cleavage) and N2/C2 (β-cleavage) are shown by jagged arrows. C2 is recognized by both 3F4 and 6H4, whereas C1 is only recognized by 6H4. B–D, lysates (15 μg) from SH-SY5Y cells expressing wtPrP were incubated in the absence or presence of PNGase F and immunoblotted with SAF32 (B), 3F4 (C), and 6H4 (25 μg of lysate) (D). Molecular mass markers (kDa) are indicated on the left.
      β-Cleavage of PrPC Is Up-regulated When Cells Are Subjected to Oxidative Stress—Since there is some evidence to suggest that β-cleavage of PrPC is mediated by ROS (
      • McMahon H.E.
      • Mange A.
      • Nishida N.
      • Creminon C.
      • Casanova D.
      • Lehmann S.
      ), we examined the effect of H2O2 and Cu2+ on the formation of the C2 fragment (Fig. 2). Changing the cell medium from serum-containing to serum-free Opti-MEM caused an increase in the production of C2 (Fig. 2, A and D) due to the removal of survival factors on withdrawal of the serum from the medium (
      • Kuwahara C.
      • Takeuchi A.M.
      • Nishimura T.
      • Haraguchi K.
      • Kubosaki A.
      • Matsumoto Y.
      • Saeki K.
      • Yokoyama T.
      • Itohara S.
      • Onodera T.
      ). In the presence of 100 μm H2O2 and 10 μm Cu2+, there was a further increase in the level of C2 above that observed in the serum-free medium-treated cells within 10 min (Fig. 2, A and D). In contrast to the changes in C2, the levels of neither full-length PrP (Fig. 2, B and E) nor the C1 fragment (Fig. 2, B and F) altered with the removal of serum or upon treatment of the cells with H2O2 and Cu2+. These observations indicate that ROS increased the production of C2 via β-cleavage but had no effect on α-cleavage. To confirm that β-cleavage of PrPC is indeed a ROS-mediated event, the effect of the hydroxyl radical quencher Me2SO was examined (Fig. 3). When cells expressing wtPrP were incubated in serum-free Opti-MEM in the presence of Me2SO, there was a dose-dependent reduction in the production of C2 (Fig. 3, A and C), consistent with β-cleavage being a ROS-mediated process.
      Figure thumbnail gr2
      FIGURE 2Copper and H2O2 up-regulate the formation of C2. SH-SY5Y cells stably expressing wtPrP were either maintained in 10% FCS-containing Dulbecco's modified Eagle's medium (Serum), maintained in Opti-MEM medium, or treated with 10 μm CuSO4 and 100 μm H2O2 in Opti-MEM for 0, 5, or 10 min, and the resulting lysates were deglycosylated with PNGase F. Immunoblot analysis of 15 μg of total cell protein was performed with either antibody 3F4 to detect C2 (A), 6H4 to detect C1 (B), or an anti-actin antibody (C). Densitometric analysis was performed on multiple immunoblots, and the results are expressed in terms of pixel intensity of the C2 (D), PrPC (E), or C1 (F) band at the period of exposure (n = 3). ***, p < 0.001.
      Figure thumbnail gr3
      FIGURE 3Formation of C2 is blocked by the hydroxyl radical scavenger Me2SO. SH-SY5Y cells expressing wtPrP were exposed to various concentrations of Me2SO for 5 h in Opti-MEM. Samples (15 μg of total protein) were digested with PNGase F before immunoblot analysis with antibody 3F4 (A) or an anti-actin antibody (B). C, multiple immunoblots were analyzed by densitometry and expressed as mean pixel intensity for the C2 fragment (n = 5).
      ROS-mediated β-Cleavage of PrPC Occurs at the Cell Surface—To determine whether cell surface PrPC was subject to ROS-mediated β-cleavage, cells expressing wtPrP were first surface-biotinylated prior to treatment with 100 μm H2O2 and 10 μm Cu2+ (Fig. 4). Following immunoprecipitation of PrP with antibody 3F4, biotinylated full-length PrP and C2 were visualized by immunoblotting with peroxidase-conjugated streptavidin. Immediately following biotinylation, negligible biotinylated C2 was detected in the cell lysate, although significant amounts of biotinylated full-length PrP were present. However, an increase in the level of biotinylated C2 fragment was clearly evident following incubation of the cells in serum-free Opti-MEM for 10 min, and this was further increased upon treatment of the cells with H2O2 and Cu2+, indicating that C2 is formed from PrPC exposed at the cell surface.
      Figure thumbnail gr4
      FIGURE 4C2 is formed at the cell surface. A, SH-SY5Y cells expressing wtPrP were surface-biotinylated for 1 h at 4 °Cand then incubated in either Opti-MEM alone or 10 μm CuSO4 and 100 μm H2O2 in Opti-MEM. Biotinylated PrP was immunoprecipitated with antibody 3F4 prior to incubation with PNGase F and then immunoblotted with peroxidase-conjugated streptavidin. B, densitometric analysis of multiple immunoblots is shown as pixel intensity of the C2 band (n = 3).
      The Octapeptide Repeats Are Required for the ROS-mediated β-Cleavage of PrPC—To determine whether ROS-mediated β-cleavage of PrPC required the octapeptide repeats, we examined the proteolytic processing of PrPΔoct that lacks the copper-binding octapeptide repeat region (
      • Perera W.S.S.
      • Hooper N.M.
      ) (Fig. 5A). Lysates from cells expressing PrPΔoct were subjected to immunoblot analysis with antibodies SAF32, 3F4, and 6H4 (Fig. 5, B–D). SAF32 failed to detect PrPΔoct, since this mutant lacks the epitope for this antibody but was detected by 3F4 and 6H4. Although antibody 6H4 clearly detected the C1 fragment in cells expressing PrPΔoct, neither antibody 6H4 nor 3F4 detected the C2 fragment. These data indicate that the octapeptide repeats are required for PrPC to undergo β-cleavage.
      Figure thumbnail gr5
      FIGURE 5Proteolytic processing of PrPC is altered in PrP mutants. A, schematic diagram of the PrP mutants used. wtPrP is shown as the mature, full-length protein with its C-terminal GPI anchor, two N-linked glycosylation sites (residues 180 and 196, lollipops), and the octapeptide repeat region (shaded). PrPΔoct lacks the entire octapeptide region, PG14 has an additional nine octapeptide repeats, and A116V has a single point mutation Ala → Val at position 116. B–D, lysates (15 μg) from SH-SY5Y cells expressing either wtPrP (lane 1), PG14 (lane 2), Δoct (lane 3), or A116V (lane 4) were digested with PNGase F and subjected to immunoblot analysis with 3F4 (B), SAF 32 (C), or 6H4 (D). Due to a lower expression level of the A116V construct, 25 μg of protein was loaded, and an increased exposure time was used to detect the protein fragments and is shown by a split in the gel. *, position of C2.
      ROS-mediated β-Cleavage Is Defective in Two Disease-associated Mutants of PrP—We examined next the proteolytic processing of two disease-associated mutants of PrP. PG14 contains an extra nine copies of the octapeptide repeat and is associated with familial human prion disease (
      • Goldfarb L.G.
      • Brown P.
      • McCombie W.R.
      • Goldgaber D.
      • Swergold G.D.
      • Wills P.R.
      • Cervenakova L.
      • Baron H.
      • Gibbs C.J.
      • Gajdusek D.C.
      ,
      • Krasemann S.
      • Zerr I.
      • Weber T.
      • Poser S.
      • Kretzschmar H.
      • Hunsmann G.
      • Bodemer W.
      ) and A116V, in which Ala116 (murine PrP numbering, equivalent to Ala117 in human PrP) is mutated to Val and is associated with Gerstmann-Sträussler-Scheinker disease (
      • 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.
      ) (Fig. 5A). Lysates from cells expressing the two mutants were subjected to immunoblot analysis with antibodies SAF32, 3F4, and 6H4 (Fig. 5, B–D). Although in cells expressing either PG14 or A116V, all three antibodies detected full-length protein, and 6H4 detected the C1 fragment, there was no detection of the C2 fragment in either cell line by antibody 3F4 or 6H4 even after prolonged exposure of the immunoblots (Fig. 5, B and D). Even upon treatment of the cells expressing PG14 or A116V with H2O2 and Cu2+ for up to 60 min, there was no evidence for the production of C2, whereas under identical conditions, C2 was clearly formed in cells expressing wtPrP (data not shown). These data indicate that in cells expressing two disease-associated mutants of PrP, although C1 is formed normally, C2 is not formed upon exposure of the cells to ROS.
      One possible explanation for the lack of ROS-mediated β-cleavage in cells expressing either PG14 or A116V is that the mutants fail to traffic to the cell surface where this processing occurs. Previously, however, we (
      • Perera W.S.S.
      • Hooper N.M.
      ) and others (
      • Lehmann S.
      • Harris D.A.
      ) have shown by surface biotinylation and immunofluorescence microscopy that PG14 is localized at the cell surface. Although the A116V mutant is expressed at a lower level than wtPrP in the SH-SY5Y cells (Fig. 6A), the amount of this mutant at the cell surface as revealed by surface biotinylation was very similar to that of wtPrP (Fig. 6B). The cell surface localization of A116V was confirmed by immunofluorescence microscopy (Fig. 6C). Like wtPrP, the A116V construct gave a similar pattern of cell surface staining, which could be abolished by incubation of the cells with bacterial phosphatidylinositol-specific phospholipase C, which cleaves the GPI anchor, releasing the protein from the membrane. Since neither the PrPΔoct nor the PG14 mutants are endocytosed when cells are exposed to Cu2+ ions (
      • Perera W.S.S.
      • Hooper N.M.
      ), we considered that the lack of β-cleavage may correlate with a deficiency in copper-mediated endocytosis. However, when cells expressing A116V were exposed to a concentration of Cu2+ ions sufficient to promote endocytosis of wtPrP (
      • Perera W.S.S.
      • Hooper N.M.
      ), this mutant was rapidly endocytosed (Fig. 6D). Thus, the inability of the A116V and PG14 mutants to reach the cell surface does not account for the lack of ROS-mediated β-cleavage of these mutants, and failure to undergo β-cleavage does not correlate with a deficiency in copper-mediated endocytosis.
      Figure thumbnail gr6
      FIGURE 6A116V is localized at the cell surface and undergoes copper-mediated endocytosis. A, lysates from SH-SY5Y cells (15 μg) expressing either wtPrP or the A116V construct were subjected to immunoblot analysis with 3F4. B, cells expressing either wtPrP or A116V were surface-biotinylated for 1 h at 4°C, and labeled PrP was immuno-precipitated with 3F4. Biotinylated PrP in the samples was detected by immunoblotting with peroxidase-conjugated streptavidin. C, immunofluorescence microscopy was performed on cells expressing either wtPrP or A116V incubated in the absence or presence of bacterial phosphatidylinositol-specific phospholipase C for 30 min at room temperature. Bar, 10 μm. D, cells expressing A116V were surface-biotinylated for 1 h at 4°C.The biotinylation reaction was quenched with 50 mm glycine in PBS before incubation of the cells in the presence or absence of 100 μm CuSO4 delivered as a histidine chelate in Opti-MEM. After 30 min, the samples were scraped into PBS or incubated with trypsin-EDTA to remove cell surface PrP. Lysates were immunoprecipitated, and biotinylated PrP was detected as described above.
      Failure to Undergo ROS-mediated β-Cleavage Correlates with a Reduced Cell Viability and Increased Levels of Intracellular Free Radicals—In order to determine whether inability to undergo ROS-mediated β-cleavage affected a biological function of PrPC, we assessed the resistance to oxidative stress of cells expressing PrPΔoct, PG14, and A116V. Previously, we have shown that SH-SY5Y cells expressing wtPrP have an increased viability, a reduced level of intracellular free radicals, and increased glutathione peroxidase activity as compared with untransfected cells upon exposure to H2O2 and Cu2+ (
      • Zeng F.
      • Watt N.T.
      • Walmsley A.R.
      • Hooper N.M.
      ). The viability of cells expressing the various mutants of PrP was assessed by measurement of cell number using Hoescht 33342 staining. Cells expressing PrPΔoct, PG14, or A116V all displayed significantly reduced viability when challenged with H2O2 and Cu2+ as compared with cells expressing wtPrP (p < 0.001) (Fig. 7A). Measurement of intracellular free radical generation in the cells was made using the fluorescent dye dihydrodichlorofluorescein diacetate in order to ascertain whether radical-mediated cell damage was altered in the cells expressing the PrP mutants. Whereas there was a significant decrease in radical formation in the wtPrP-expressing cells as compared with the untransfected cells, cells expressing PrPΔoct, PG14, or A116V had a level of radical formation similar to that of the untransfected cells (Fig. 7B). Glutathione peroxidase is a key component of an important antioxidant pathway in neurons, detoxifying H2O2 upon glutathione oxidation. The wtPrP-expressing cells had a higher level of glutathione peroxidase activity than the untransfected cells, whereas the cells expressing PrPΔoct, PG14, or A116V all had significantly reduced glutathione peroxidase activity as compared with wtPrP-expressing cells (p < 0.001) (Fig. 7C). Together, these data indicate that cells expressing PrPΔoct, PG14, or A116V, none of which undergo ROS-mediated β-cleavage, were not protected against oxidative stress in the same way that cells expressing wtPrP were protected.
      Figure thumbnail gr7
      FIGURE 7Reduced viability and glutathione peroxidase activity and increased radical generation in cells expressing mutants of PrP unable to produce C2. A, Hoescht 33342 staining of untransfected SH-SY5Y (Un) cells or cells containing either wtPrP or the indicated mutants exposed to 8 μm CuSO4 and 100 μm H2O2 for 48 h, expressed as a percentage of control, untreated staining for each individual construct (n = 8). B, measurement of intracellular radical generation using dihydrodichlorofluorescein diacetate in cells treated with 8 μm CuSO4 and 100 μm H2O2 for 4 h. Data are expressed as percentage of control, untreated staining for each individual construct (n = 8). C, glutathione peroxidase activity in untransfected SH-SY5Y cells or cells containing the indicated constructs was determined as described under “Experimental Procedures.” Results are shown as change in absorbance/ml/mg protein. ***, p < 0.001.
      To further examine the relationship between ROS-mediated β-cleavage and the resistance of cells to oxidative stress, we sought to block β-cleavage of wtPrP and then assess cell viability upon exposure to ROS. We reasoned that antibody SAF32, which binds to the octapeptide repeats may prevent β-cleavage. Cells expressing wtPrP were incubated in the presence of either antibody SAF32 or antibody 3F4 as control prior to exposure to Cu2+ and H2O2 (Fig. 8). Although the formation of C2 still occurred in cells incubated with 3F4, its production was significantly reduced by SAF32 (Fig. 8, A and C). Furthermore, cells incubated with SAF32 had a significantly lower viability when exposed to Cu2+ and H2O2 than cells exposed to either 3F4 or no antibody (Fig. 8D), providing further evidence that β-cleavage is involved in the cellular response to oxidative stress.
      Figure thumbnail gr8
      FIGURE 8Binding of an antibody to the octapeptide repeats blocks β-cleavage and compromises cell viability. A, SH-SY5Y cells expressing wtPrP were incubated with either antibody 3F4 or SAF32 at the indicated concentrations in Opti-MEM for 5 h. The cell lysates were deglycosylated with PNGase F and immunoblotted with either antibody 3F4 to ascertain the C2 levels (A) or an anti-actin antibody (B). C, densitometric analysis of multiple immunoblots is shown as pixel intensity of the C2 band as a percentage of the C2 band produced in the cells not incubated with antibody (n = 3). D, Hoescht 33342 staining of SH-SY5Y cells containing wtPrP exposed to 8 μm CuSO4 and 100 μm H2O2 for 5 h in the absence or presence of 10 μg of 3F4 or 10 μg of SAF32 antibody as indicated. Data are expressed as a percentage of staining in the absence of Cu2+ and H2O2 for each condition (n = 8). ***, p < 0.001.
      C2 Formed from the ROS-mediated β-Cleavage of PrPC Is neither Proteinase K-resistant nor Generated by Calpains—Recently, it has been reported that in scrapie-infected mouse brain and cells, a C2-like fragment is present that is proteinase K-resistant and is generated through cleavage of PrPSc by calpains (
      • Yadavalli R.
      • Guttmann R.P.
      • Seward T.
      • Centers A.P.
      • Williamson R.A.
      • Telling G.C.
      ). In contrast, the C2 fragment generated from wtPrP expressed in the SH-SY5Y cells in the present study was completely proteinase K-sensitive (Fig. 9A). We examined also whether the calpain inhibitor ALLM could block the formation of C2 (Fig. 9, B and D). Interestingly, incubation of the cells with 50 μm ALLM dissolved in Me2SO led to a reduction in the formation of C2. However, this was due to the free radical quenching effect of Me2SO, since Me2SO alone blocked the formation of C2 (Fig. 9, B and D; see Fig. 3), and ALLM dissolved in ethanol did not inhibit the formation of C2, despite the fact that ALLM (at 10 μm) dissolved in either Me2SO or ethanol completely inhibited the activity of recombinant calpain against a fluorimetric peptide substrate (Fig. 9E). Thus, the calpain inhibitor ALLM failed to block the ROS-mediated β-cleavage of PrPC in the SH-SY5Y cells, and the resulting C2 fragment was sensitive to proteinase K digestion.
      Figure thumbnail gr9
      FIGURE 9C2 is proteinase K sensitive, and its formation is not blocked by calpain inhibitors. A, lysates (15 μg of total protein) from cells exposed to 10 μm CuSO4 and 100 μm H2O2 were incubated in the presence or absence of PNGase F followed by digestion with 5 μg/ml proteinase K (PK) and immunoblotted with antibody 3F4. B and C, SH-SY5Y cells expressing wtPrP were incubated in the presence or absence of the calpain inhibitor 50 μm ALLM for 5 h using either Me2SO or ethanol as carrier. Samples (15 μg) were digested with PNGase F before immunoblot analysis using antibody 3F4 (B) or an anti-actin antibody (C). D, densitometric analysis of multiple immunoblots is shown as pixel intensity of the C2 band (n = 3). E, recombinant calpain was assayed with a fluorimetric substrate in the absence or presence of 10 μm ALLM in either Me2SO or EtOH. Activity is represented as percentage of the activity in the absence of inhibitor and carrier (n = 2). ***, p < 0.001.

      DISCUSSION

      Like many proteins, PrPC is subject to a variety of proteolytic cleavage events that may modulate its biological functions. However, the role of these cleavages and the function of the generated fragments remain to be determined. In the present study, we show for the first time that the ROS-mediated β-cleavage of PrPC occurs at the cell surface, requires the octapeptide repeat region within PrPC, and is defective in two forms of PrP associated with prion disease. Crucially, we show that this failure to undergo ROS-mediated β-cleavage correlates with an increased sensitivity of cells to oxidative stress, providing the first direct evidence that β-cleavage may be a critical first step in the mechanism whereby PrPC protects cells against oxidative stress.
      Although the β-cleavage of PrPC at the C-terminal end of the octapeptide repeats, near residue 90, has been observed by several groups (
      • Pan K.-M.
      • Stahl N.
      • Prusiner S.B.
      ,
      • Jimenez-Huete A.
      • Lievens P.M.J.
      • Vidal R.
      • Piccardo P.
      • Ghetti B.
      • Tagliavini F.
      • Frangione B.
      • Prelli F.
      ,
      • Taraboulos A.
      • Raeber A.J.
      • Borchelt D.R.
      • Serban D.
      • Prusiner S.B.
      ), the mechanism of this cleavage remained elusive until McMahon et al. (
      • McMahon H.E.
      • Mange A.
      • Nishida N.
      • Creminon C.
      • Casanova D.
      • Lehmann S.
      ) reported that it was ROS-mediated and Cu2+-dependent. These authors showed that soluble PrP in conditioned medium underwent β-cleavage upon exposure to millimolar concentrations of H2O2 in the presence of 10 μm Cu2+. We have extended this observation to a more in vivo setting by showing that exposure of intact cells expressing PrPC to micromolar concentrations of H2O2 and Cu2+stimulates β-cleavage, as evidenced by the formation of the C-terminal fragment C2. It has been hypothesized that PrPC is involved in the cellular response mechanism to external oxidative stress, possibly acting as a sensor of ROS (
      • Brown D.R.
      ,
      • Milhavet O.
      • Lehmann S.
      ). Although it has previously been shown that ROS causes β-cleavage of soluble PrP (
      • McMahon H.E.
      • Mange A.
      • Nishida N.
      • Creminon C.
      • Casanova D.
      • Lehmann S.
      ), it has not been investigated whether the β-cleavage of PrPC may be involved in the mechanism by which cells respond to oxidative stress. Our observations that surface-biotinylated PrPC rapidly (within minutes) undergoes β-cleavage upon exposure of cells to ROS and that lack of β-cleavage correlates with an increased sensitivity to oxidative stress provides the first evidence that this processing event is an early step in the cellular response to external oxidative stress.
      Recently, it has been reported that in scrapie-infected mouse brain and in persistently infected scrapie mouse brain cells, a C2-like fragment is present that is proteinase K-resistant and is generated through cleavage of PrPSc by calpains (
      • Yadavalli R.
      • Guttmann R.P.
      • Seward T.
      • Centers A.P.
      • Williamson R.A.
      • Telling G.C.
      ). The formation of C2 from PrPC in the SH-SY5Y cells was not blocked by a specific calpain inhibitor, nor was C2 proteinase K resistant, consistent with the study of Yadavalli et al. (
      • Yadavalli R.
      • Guttmann R.P.
      • Seward T.
      • Centers A.P.
      • Williamson R.A.
      • Telling G.C.
      ) that in uninfected cells and brain tissue the observed C2 fragment is not proteinase K-resistant. Thus, it appears that PrPC is subject to ROS-mediated β-cleavage, which produces the C2 fragment that is proteinase K-sensitive, whereas PrPSc is cleaved by calpain to produce a C2-like fragment that is proteinase K-resistant. It is plausible that the conformational change from PrPC to PrPSc exposes a cleavage site for calpain that is not accessible in PrPC.
      Through the Fenton reaction, copper (and iron) promote the formation of ROS such as the hydroxyl radical (·OH) from H2O2, which, although itself not a ROS, is an important mediator of oxidative stress in neurons (
      • White A.R.
      • Collins S.J.
      • Maher F.
      • Jobling M.F.
      • Stewart L.R.
      • Thyer J.M.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ). When the metal is protein-bound, since the Cu2+ ions are in the octapeptide repeats of PrPC, the oxidative-reduction reaction can locally generate ROS that may react at specific sites in the protein, possibly resulting in peptide bond cleavage (
      • Kim K.
      • Rhee S.G.
      • Stadtman E.R.
      ). The critical involvement of ROS in the β-cleavage of PrPC is further evidenced by the inhibitory effect of the hydroxyl radical trapping agent Me2SO. The importance of the Cu2+ ions bound at the octapeptide repeats of PrPC contributing to the ROS-mediated β-cleavage is evidenced by the lack of ROS-mediated cleavage of PrPΔoct, which lacks the octapeptide repeats and therefore has no Cu2+ ions bound in this region of the protein. Although Cu2+ binding sites downstream of the octapeptide repeats have been identified (
      • Jackson G.S.
      • Murray I.
      • Hosszu L.L.
      • Gibbs N.
      • Waltho J.P.
      • Clarke A.R.
      • Collinge J.
      ,
      • Qin K.
      • Yang Y.
      • Mastrangelo P.
      • Westaway D.
      ,
      • Burns C.S.
      • Aronoff-Spencer E.
      • Legname G.
      • Prusiner S.B.
      • Antholine W.E.
      • Gerfen G.J.
      • Peisach J.
      • Millhauser G.L.
      ), the Cu2+ bound at these sites does not appear to be involved in the ROS-mediated cleavage of PrPC, as evidenced by the lack of cleavage of PrPΔoct, which retains these down-stream Cu2+ binding sites.
      Collectively, our data indicate that ROS-mediated β-cleavage of PrPC may be the first step in a cascade of cellular events that lead the cell to mount a response to increased oxidative stress. Consistent with this is the observation that cells expressing wtPrP have increased viability and glutathione peroxidase activity and reduced intracellular free radicals when exposed to ROS as compared with untransfected cells and that such protective responses to ROS are not observed in the cells expressing PrPΔoct, which fails to undergo β-cleavage due to the lack of the octapeptide repeats or in cells expressing wtPrP when β-cleavage is blocked by the binding of antibody SAF32 to the octapeptide repeats.
      At first sight, it appears somewhat surprising that neither PG14 nor A116V was subject to ROS-mediated β-cleavage. As shown in the present study (for A116V) and elsewhere (for PG14) (
      • Perera W.S.S.
      • Hooper N.M.
      ,
      • Lehmann S.
      • Harris D.A.
      ), this is not due to a failure of these mutants of PrP to reach the cell surface, where ROS-mediated β-cleavage occurs. Furthermore, it is not linked to an inability to undergo copper-mediated endocytosis as seen with both PG14 and PrPΔoct (
      • Perera W.S.S.
      • Hooper N.M.
      ), since A116V was efficiently endocytosed upon incubation of the cells with copper. PG14, which contains an extra nine copies of the octapeptide repeat, might, if anything, be expected to be more susceptible to copper-dependent ROS-mediated cleavage. Clearly, this is not the case, and the extended octapeptide repeat region, possibly through the formation of an altered relatively proteinase-resistant structure (
      • Lehmann S.
      • Harris D.A.
      ,
      • Chiesa R.
      • Drisaldi B.
      • Quaglio E.
      • Migheli A.
      • Piccardo P.
      • Ghetti B.
      • Harris D.A.
      ), may somehow prevent ROS-mediated β-cleavage. In the case of A116V, why a single conservative point mutation some 25 residues away from the site of β-cleavage has such a dramatic effect is not immediately obvious. One possibility is that this mutation disrupts the folding of the polypeptide chain and thus prevents ROS-mediated β-cleavage occurring. In this context, it is interesting to note that mutation of Ala113, Ala115, and Ala118 to valines enhances the folding of peptides spanning this region into compact structural units, significantly enhancing the formation of extensive β-sheet fibrils (
      • Inouye H.
      • Bond J.
      • Baldwin M.A.
      • Ball H.L.
      • Prusiner S.B.
      • Kirschner D.A.
      ).
      The results of the present study do not allow us to directly address which of the proteolytic fragments, N2 or C2, produced from ROS-mediated β-cleavage of PrPC is responsible for propagating the survival signal. The soluble N2 fragment may act as a signaling molecule analogous to peptide growth factors (
      • Brown D.R.
      ). In support of this is the observation that deletion of the N-terminal residues 23–88 from PrP abrogates the potential to rescue PrP-deficient mice from Doppel-induced neurodegeneration (
      • Atarashi R.
      • Nishida N.
      • Shigematsu K.
      • Goto S.
      • Kondo T.
      • Sakaguchi S.
      • Katamine S.
      ) and that cells expressing a construct of PrP in which the N terminus is tethered to the membrane through an uncleaved signal peptide/transmembrane anchor are severely compromised in their resistance to oxidative stress (
      • Zeng F.
      • Watt N.T.
      • Walmsley A.R.
      • Hooper N.M.
      ). Alternatively, it has been suggested that the GPI-anchored C2 fragment is important in mediating the cellular response to oxidative stress via dimerization and activation of signal transduction pathways (
      • Milhavet O.
      • Lehmann S.
      ) and that the protective function of C2 is turned off by subsequent α-cleavage to generate C1 (
      • Mange A.
      • Beranger F.
      • Peoc'h K.
      • Onodera T.
      • Frobert Y.
      • Lehmann S.
      ). Thus, there is a precursor-product relationship between the C2 and C1 fragments. However, our data would argue against such a relationship, because although PrPΔoct, PG14, and A116V all failed to produce C2, they all produced amounts of C1 similar to those produced by wtPrP. Rather, we favor the scenario that the α- and β-cleavages of PrPC are independent proteolytic events similar to the α- and β-cleavages of the Alzheimer's amyloid precursor protein (
      • Hooper N.M.
      • Turner A.J.
      ).
      The observation that ROS-mediated β-cleavage of PrPC is defective in the two disease-associated mutants PG14 and A116V adds further weight to the argument that prion diseases are, in part, due to the loss of a normal function of PrPC (
      • Hetz C.
      • Maundrell K.
      • Soto C.
      ). Clearly, the inability of cells expressing the disease-associated PG14 and A116V mutants to mount a protective response against oxidative stress would be detrimental. There is increasing evidence that oxidative stress is involved in prion diseases (
      • Guentchev M.
      • Voigtlander T.
      • Haberler C.
      • Groschup M.H.
      • Budka H.
      ,
      • Lee D.W.
      • Sohn H.O.
      • Lim H.B.
      • Lee Y.G.
      • Kim Y.S.
      • Carp R.I.
      • Wisniewski H.M.
      ,
      • Choi S.I.
      • Ju W.K.
      • Choi E.K.
      • Kim J.
      • Lea H.Z.
      • Carp R.I.
      • Wisniewski H.M.
      • Kim Y.S.
      ). This could come about because, upon conversion to PrPSc, PrPC is no longer available to be subject to ROS-mediated β-cleavage as part, possibly the first step, of the cellular mechanism to protect against oxidative stress. In cases of prion disease due to mutation in PrP, such as in PG14 and A116V, that prevent ROS-mediated β-cleavage of PrPC, the normal cellular response to oxidative stress is compromised, and this in turn may contribute to the neurodegeneration observed.

      References

        • Prusiner S.B.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383
        • Bueler H.
        • Aguzzi A.
        • Sailer A.
        • Greiner R.A.
        • Autenried P.
        • Aguet M.
        • Weissmann C.
        Cell. 1993; 73: 1339-1347
        • Prusiner S.B.
        • Groth D.
        • Serban A.
        • Koehler R.
        • Foster D.
        • Torchia M.
        • Burton D.
        • Yang S.L.
        • DeArmond S.J.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10608-10612
        • Hetz C.
        • Maundrell K.
        • Soto C.
        Trends Mol. Med. 2003; 9: 237-243
        • Yokoyama T.
        • Kimura K.M.
        • Ushiki Y.
        • Yamada S.
        • Morooka A.
        • Nakashiba T.
        • Sassa T.
        • Itohara S.
        J. Biol. Chem. 2001; 276: 11265-11271
        • Vassallo N.
        • Herms J.
        J. Neurochem. 2003; 86: 538-544
        • Roucou X.
        • Gains M.
        • LeBlanc A.C.
        J. Neurosci. Res. 2004; 75: 153-161
        • Brown D.R.
        • Qin K.
        • Herms J.W.
        • Madlung A.
        • Manson J.
        • Strome R.
        • Fraser P.E.
        • Kruck T.
        • von Bohlen A.
        • Schulz-Schaeffer W.
        • Giese A.
        • Westaway D.
        • Kretzschmar H.
        Nature. 1997; 390: 684-687
        • Miura T.
        • Hori-i A.
        • Mototani H.
        • Takeuchi H.
        Biochemistry. 1999; 38: 11560-11569
        • Viles J.H.
        • Cohen F.E.
        • Prusiner S.B.
        • Goodin D.B.
        • Wright P.E.
        • Dyson H.J.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047
        • Pauly P.C.
        • Harris D.A.
        J. Biol. Chem. 1998; 273: 33107-33110
        • Perera W.S.S.
        • Hooper N.M.
        Curr. Biol. 2001; 11: 519-523
        • Brown D.R.
        J. Neurochem. 2003; 87: 377-385
        • Brown D.R.
        • Schulz-Schaeffer W.J.
        • Schmidt B.
        • Kretzschmar H.A.
        Exp. Neurol. 1997; 146: 104-112
        • Kuwahara C.
        • Takeuchi A.M.
        • Nishimura T.
        • Haraguchi K.
        • Kubosaki A.
        • Matsumoto Y.
        • Saeki K.
        • Yokoyama T.
        • Itohara S.
        • Onodera T.
        Nature. 1999; 400: 225-226
        • White A.R.
        • Collins S.J.
        • Maher F.
        • Jobling M.F.
        • Stewart L.R.
        • Thyer J.M.
        • Beyreuther K.
        • Masters C.L.
        • Cappai R.
        Am. J. Pathol. 1999; 155: 1723-1730
        • Zeng F.
        • Watt N.T.
        • Walmsley A.R.
        • Hooper N.M.
        J. Neurochem. 2003; 84: 480-490
        • Pan K.-M.
        • Stahl N.
        • Prusiner S.B.
        Protein Sci. 1992; 1: 1343-1352
        • Shyng S.-L.
        • Huber M.T.
        • Harris D.A.
        J. Biol. Chem. 1993; 268: 15922-15928
        • Chen S.G.
        • Teplow D.B.
        • Parchi P.
        • Teller J.K.
        • Gambetti P.
        • Autilio-Gambetti L.
        J. Biol. Chem. 1995; 270: 19173-19180
        • Jimenez-Huete A.
        • Lievens P.M.J.
        • Vidal R.
        • Piccardo P.
        • Ghetti B.
        • Tagliavini F.
        • Frangione B.
        • Prelli F.
        Am. J. Pathol. 1998; 153: 1561-1572
        • Mange A.
        • Beranger F.
        • Peoc'h K.
        • Onodera T.
        • Frobert Y.
        • Lehmann S.
        Biol. Cell. 2004; 96: 125-132
        • Vincent B.
        • Paitel E.
        • Saftig P.
        • Frobert Y.
        • Hartmann D.
        • De Strooper B.
        • Grassi J.
        • Lopez-Perez E.
        • Checler F.
        J. Biol. Chem. 2001; 276: 37743-37746
        • Taraboulos A.
        • Raeber A.J.
        • Borchelt D.R.
        • Serban D.
        • Prusiner S.B.
        Mol. Biol. Cell. 1992; 3: 851-863
        • McMahon H.E.
        • Mange A.
        • Nishida N.
        • Creminon C.
        • Casanova D.
        • Lehmann S.
        J. Biol. Chem. 2001; 276: 2286-2291
        • Parkin E.T.
        • Watt N.T.
        • Turner A.J.
        • Hooper N.M.
        J. Biol. Chem. 2004; 279: 11170-11178
        • Walmsley A.R.
        • Zeng F.
        • Hooper N.M.
        EMBO J. 2001; 20: 703-712
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke B.J.
        • Olson B.J.
        • Klenk D.C.
        Anal. Biochem. 1985; 150: 76-85
        • Goldfarb L.G.
        • Brown P.
        • McCombie W.R.
        • Goldgaber D.
        • Swergold G.D.
        • Wills P.R.
        • Cervenakova L.
        • Baron H.
        • Gibbs C.J.
        • Gajdusek D.C.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10926-10930
        • Krasemann S.
        • Zerr I.
        • Weber T.
        • Poser S.
        • Kretzschmar H.
        • Hunsmann G.
        • Bodemer W.
        Brain Res. Mol. Brain Res. 1995; 34: 173-176
        • 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.
        Science. 1998; 279: 827-834
        • Lehmann S.
        • Harris D.A.
        J. Biol. Chem. 1995; 270: 24589-24597
        • Yadavalli R.
        • Guttmann R.P.
        • Seward T.
        • Centers A.P.
        • Williamson R.A.
        • Telling G.C.
        J. Biol. Chem. 2004; 279: 21948-21956
        • Brown D.R.
        Trends Neurosci. 2001; 24: 85-90
        • Milhavet O.
        • Lehmann S.
        Brain Res. Brain Res. Rev. 2002; 38: 328-339
        • Kim K.
        • Rhee S.G.
        • Stadtman E.R.
        J. Biol. Chem. 1985; 260: 15394-15397
        • Jackson G.S.
        • Murray I.
        • Hosszu L.L.
        • Gibbs N.
        • Waltho J.P.
        • Clarke A.R.
        • Collinge J.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8531-8535
        • Qin K.
        • Yang Y.
        • Mastrangelo P.
        • Westaway D.
        J. Biol. Chem. 2002; 277: 1981-1990
        • Burns C.S.
        • Aronoff-Spencer E.
        • Legname G.
        • Prusiner S.B.
        • Antholine W.E.
        • Gerfen G.J.
        • Peisach J.
        • Millhauser G.L.
        Biochemistry. 2003; 42: 6794-6803
        • Lehmann S.
        • Harris D.A.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5610-5614
        • Chiesa R.
        • Drisaldi B.
        • Quaglio E.
        • Migheli A.
        • Piccardo P.
        • Ghetti B.
        • Harris D.A.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5574-5579
        • Inouye H.
        • Bond J.
        • Baldwin M.A.
        • Ball H.L.
        • Prusiner S.B.
        • Kirschner D.A.
        J. Mol. Biol. 2000; 300: 1283-1296
        • Atarashi R.
        • Nishida N.
        • Shigematsu K.
        • Goto S.
        • Kondo T.
        • Sakaguchi S.
        • Katamine S.
        J. Biol. Chem. 2003; 278: 28944-28949
        • Hooper N.M.
        • Turner A.J.
        Curr. Med. Chem. 2002; 9: 1107-1119
        • Guentchev M.
        • Voigtlander T.
        • Haberler C.
        • Groschup M.H.
        • Budka H.
        Neurobiol. Dis. 2000; 7: 270-273
        • Lee D.W.
        • Sohn H.O.
        • Lim H.B.
        • Lee Y.G.
        • Kim Y.S.
        • Carp R.I.
        • Wisniewski H.M.
        Free Radic. Res. 1999; 30: 499-507
        • Choi S.I.
        • Ju W.K.
        • Choi E.K.
        • Kim J.
        • Lea H.Z.
        • Carp R.I.
        • Wisniewski H.M.
        • Kim Y.S.
        Acta Neuropathol. (Berl.). 1998; 96: 279-286