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• W2013618043 abstract "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. 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 (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5099) Google Scholar). In prion diseases, the normal cellular form of the prion protein (PrPC) 4The abbreviations used are:PrPCcellular form of the prion proteinPrPScinfectious, protease resistant form of PrPALLMN-acetyl-Leu-Leu-Met-aldehydeFCSfetal calf serumPBSphosphate-buffered salinePNGase FPeptide:N-glycosidase FROSreactive oxygen specieswtPrPwild type PrPGPIglycosylphosphatidylinositol 4The abbreviations used are:PrPCcellular form of the prion proteinPrPScinfectious, protease resistant form of PrPALLMN-acetyl-Leu-Leu-Met-aldehydeFCSfetal calf serumPBSphosphate-buffered salinePNGase FPeptide:N-glycosidase FROSreactive oxygen specieswtPrPwild type PrPGPIglycosylphosphatidylinositol 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 (2Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1802) Google Scholar, 3Prusiner 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: 10608-10612Crossref PubMed Scopus (416) Google Scholar), 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 (4Hetz C. Maundrell K. Soto C. Trends Mol. Med. 2003; 9: 237-243Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 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 (5Yokoyama T. Kimura K.M. Ushiki Y. Yamada S. Morooka A. Nakashiba T. Sassa T. Itohara S. J. Biol. Chem. 2001; 276: 11265-11271Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). cellular form of the prion protein infectious, protease resistant form of PrP N-acetyl-Leu-Leu-Met-aldehyde fetal calf serum phosphate-buffered saline Peptide:N-glycosidase F reactive oxygen species wild type PrP glycosylphosphatidylinositol cellular form of the prion protein infectious, protease resistant form of PrP N-acetyl-Leu-Leu-Met-aldehyde fetal calf serum phosphate-buffered saline Peptide:N-glycosidase F reactive oxygen species wild type PrP glycosylphosphatidylinositol Among the neuroprotective functions of PrPC are roles in copper homeostasis and the cellular resistance to oxidative stress (6Vassallo N. Herms J. J. Neurochem. 2003; 86: 538-544Crossref PubMed Scopus (175) Google Scholar, 7Roucou X. Gains M. LeBlanc A.C. J. Neurosci. Res. 2004; 75: 153-161Crossref PubMed Scopus (145) Google Scholar). PrPC binds Cu2+ ions, primarily within the N-terminal octapeptide repeats (8Brown 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: 684-687Crossref PubMed Scopus (37) Google Scholar, 9Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (248) Google Scholar, 10Viles 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: 2042-2047Crossref PubMed Scopus (506) Google Scholar), undergoes endocytosis upon exposure of cells to Cu2+ (11Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and modulates neuronal Cu2+ content (13Brown D.R. J. Neurochem. 2003; 87: 377-385Crossref PubMed Scopus (45) Google Scholar), 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) (14Brown D.R. Schulz-Schaeffer W.J. Schmidt B. Kretzschmar H.A. Exp. Neurol. 1997; 146: 104-112Crossref PubMed Scopus (390) Google Scholar, 15Kuwahara C. Takeuchi A.M. Nishimura T. Haraguchi K. Kubosaki A. Matsumoto Y. Saeki K. Yokoyama T. Itohara S. Onodera T. Nature. 1999; 400: 225-226Crossref PubMed Scopus (373) Google Scholar, 16White 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: 1723-1730Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Zeng F. Watt N.T. Walmsley A.R. Hooper N.M. J. Neurochem. 2003; 84: 480-490Crossref PubMed Scopus (67) Google Scholar), 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 (18Pan K.-M. Stahl N. Prusiner S.B. Protein Sci. 1992; 1: 1343-1352Crossref PubMed Scopus (172) Google Scholar, 19Shyng S.-L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar, 20Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 21Jimenez-Huete A. Lievens P.M.J. Vidal R. Piccardo P. Ghetti B. Tagliavini F. Frangione B. Prelli F. Am. J. Pathol. 1998; 153: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This processing has been referred to as α-cleavage (22Mange A. Beranger F. Peoc'h K. Onodera T. Frobert Y. Lehmann S. Biol. Cell. 2004; 96: 125-132Crossref PubMed Scopus (138) Google Scholar) and may be mediated by ADAM 10 and ADAM 17, members of the ADAM (adisintegrin and metalloprotease) family (23Vincent B. Paitel E. Saftig P. Frobert Y. Hartmann D. De Strooper B. Grassi J. Lopez-Perez E. Checler F. J. Biol. Chem. 2001; 276: 37743-37746Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 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 (18Pan K.-M. Stahl N. Prusiner S.B. Protein Sci. 1992; 1: 1343-1352Crossref PubMed Scopus (172) Google Scholar, 21Jimenez-Huete A. Lievens P.M.J. Vidal R. Piccardo P. Ghetti B. Tagliavini F. Frangione B. Prelli F. Am. J. Pathol. 1998; 153: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 24Taraboulos A. Raeber A.J. Borchelt D.R. Serban D. Prusiner S.B. Mol. Biol. Cell. 1992; 3: 851-863Crossref PubMed Scopus (237) Google Scholar). This cleavage event appears to be mediated by ROS (25McMahon H.E. Mange A. Nishida N. Creminon C. Casanova D. Lehmann S. J. Biol. Chem. 2001; 276: 2286-2291Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and has been termed β-cleavage (22Mange A. Beranger F. Peoc'h K. Onodera T. Frobert Y. Lehmann S. Biol. Cell. 2004; 96: 125-132Crossref PubMed Scopus (138) Google Scholar). 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 (26Parkin E.T. Watt N.T. Turner A.J. Hooper N.M. J. Biol. Chem. 2004; 279: 11170-11178Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). 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. cDNA Constructs and Cell Culture—The construction of wtPrP, PrPΔoct, and PG14 in pIRESneo has been described previously (12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). 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 (27Walmsley A.R. Zeng F. Hooper N.M. EMBO J. 2001; 20: 703-712Crossref PubMed Scopus (67) Google Scholar). 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 (28Smith 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: 76-85Crossref PubMed Scopus (18489) Google Scholar). 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 (12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). 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 (27Walmsley A.R. Zeng F. Hooper N.M. EMBO J. 2001; 20: 703-712Crossref PubMed Scopus (67) Google Scholar). 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 (17Zeng F. Watt N.T. Walmsley A.R. Hooper N.M. J. Neurochem. 2003; 84: 480-490Crossref PubMed Scopus (67) Google Scholar). 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 (17Zeng F. Watt N.T. Walmsley A.R. Hooper N.M. J. Neurochem. 2003; 84: 480-490Crossref PubMed Scopus (67) Google Scholar). 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. 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) (20Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 21Jimenez-Huete A. Lievens P.M.J. Vidal R. Piccardo P. Ghetti B. Tagliavini F. Frangione B. Prelli F. Am. J. Pathol. 1998; 153: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). 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 (19Shyng S.-L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar, 20Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 21Jimenez-Huete A. Lievens P.M.J. Vidal R. Piccardo P. Ghetti B. Tagliavini F. Frangione B. Prelli F. Am. J. Pathol. 1998; 153: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Mange A. Beranger F. Peoc'h K. Onodera T. Frobert Y. Lehmann S. Biol. Cell. 2004; 96: 125-132Crossref PubMed Scopus (138) Google Scholar). 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 (25McMahon H.E. Mange A. Nishida N. Creminon C. Casanova D. Lehmann S. J. Biol. Chem. 2001; 276: 2286-2291Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), 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 (15Kuwahara C. Takeuchi A.M. Nishimura T. Haraguchi K. Kubosaki A. Matsumoto Y. Saeki K. Yokoyama T. Itohara S. Onodera T. Nature. 1999; 400: 225-226Crossref PubMed Scopus (373) Google Scholar). 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 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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 (12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) (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. 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 (29Goldfarb 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: 10926-10930Crossref PubMed Scopus (288) Google Scholar, 30Krasemann S. Zerr I. Weber T. Poser S. Kretzschmar H. Hunsmann G. Bodemer W. Brain Res. Mol. Brain Res. 1995; 34: 173-176Crossref PubMed Scopus (89) Google Scholar) 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 (31Hegde 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: 827-834Crossref PubMed Scopus (614) Google Scholar) (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 (12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) and others (32Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) 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 (12Perera W.S.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), 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 endocyt" @default.
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• W2013618043 title "Reactive Oxygen Species-mediated β-Cleavage of the Prion Protein in the Cellular Response to Oxidative Stress" @default.
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