Physiological Role of the Cellular Prion Protein - American Chemical

Physiological Role of the Cellular Prion Protein (PrPc): Protein Profiling Study in Two Cell Culture Systems Sanja Ramljak,†,# Abdul R. Asif,‡,# Victor W. Armstrong,‡ Arne Wrede,§ Martin H. Groschup,| Anne Buschmann,| Walter Schulz-Schaeffer,§ Walter Bodemer,⊥ and Inga Zerr*,† Department of Neurology, Georg-August University, Go¨ttingen, Germany, Department of Clinical Chemistry, Georg-August University, Go¨ttingen, Germany, Department of Neuropathology, Georg-August University, Go¨ttingen, Germany, Friedrich-Loeffler-Institut, INEID, Greifswald-Insel Riems, Germany, and Department of Infectious Pathology, German Primate Center, Go¨ttingen, Germany Received November 8, 2007

The physiological role of the cellular prion protein (PrPc) is still not fully understood. Current evidence strongly suggests that PrPc overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrPc in PrPc-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/ or the level of PrPc expression seem to be critical for the fluctuation between PrPc’s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrPc itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrPc into PrPc-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrPc-deficient cells) endogenous PrPc. Protein profiling following transient PrPc overexpression in HEK 293 cells revealed a major PrPc involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrPc into mouse neuronal PrPc-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrPc overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrPc expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrPc in cell physiology. Keywords: cellular prion protein • function • proteome • human embryonic kidney 293 cells • prion protein deficient cells

Introduction Because of the cellular prion protein’s (PrPc) involvement in different cellular processes, such as the regulation of cell death,1–3 the protection against oxidative stress,4,5 copper binding6,7 and the modulation of several signal transduction pathways known to promote cellular survival,8–10 the overall picture regarding its actual physiological function remains ambiguous. However, PrPc’s role in the regulation of cell death is undisputed. An introduction of PrPc into PrPc-deficient hippocampal neurons undergoing apoptosis rescued the apo* Correspondence should be addressed to Prof. Dr. Inga Zerr, Dept. of Neurology, Georg-August University, Go¨ttingen, Robert-Koch-Str. 40, 37075 Go¨ttingen, Germany. Tel: ++49 551 39 6636. Fax: + +49 551 39 7020. E-mail: [email protected]. † Department of Neurology, Georg-August University. ‡ Department of Clinical Chemistry, Georg-August University. # These authors contributed equally to this work. § Department of Neuropathology, Georg-August University. | Friedrich-Loeffler-Institut, INEID. ⊥ Department of Infectious Pathology, German Primate Center. 10.1021/pr7007187 CCC: $40.75

 2008 American Chemical Society

ptotic phenotype.1 Moreover, in vivo studies demonstrated that following acute seizures or an ischemic injury, physiological levels of PrPc exert a neuroprotective effect.11–13 Conversely, the overproduction of PrPc in different cell lines appears to sensitize cells to apoptosis through the control of p53 activity3 and to have a detrimental effect in vivo.14,15 Therefore, the levels of PrPc expression seem to be decisive for fluctuation between pro- and antiapoptotic phenotype. Genomic-based differential display methodology was used to investigate the function of PrPc.16–18 However, transcription does not always result in the formation of functional proteins. Thus, it is essential to directly uncover proteins whose expression is modulated through PrPc. As yet, a proteome approach was rarely used for studying the physiological role of PrPc.19,20 To reveal the proteome changes caused by the presence/ absence of endogenous PrPc and/or different PrPc expression levels, we have chosen two approaches: transient overexpression of human PrPc in HEK 293 cells, expressing endogenous PrPc, and transient introduction of the human prion protein Journal of Proteome Research 2008, 7, 2681–2695 2681 Published on Web 06/07/2008

research articles gene (PRNP) into mouse neuronal prion protein deficient (Prnp0/0) cells. The proteome within each cell group was compared and the proteomes between the cell groups were screened for unique and common patterns in protein expression. We reasoned that the presence/absence and/or different PrPc expression levels in both cell systems would induce specific protein expression patterns that either directly or indirectly contribute to previously observed pro- and antiapoptotic phenotypes. This again might improve our understanding not only of PrPc function, but also of the molecular mechanisms underlying the pathogenesis of prion diseases.

Materials and Methods Cloning Procedure: Polymerase Chain Reaction (PCR). DNA was extracted from 100 µL of fresh, anticoagulated whole human blood with a commercially available kit (Qiagen, Heiden, Germany). PCR was carried out in a volume of 50 µL containing 1 U Thermopol Vent polymerase (2000 U/mL) (New England Biolabs, Frankfurt am Main, Germany), 20 pmol of each oligonucleotide primer, 20 mM dNTPs, 2 mM MgSO4, 10× buffer for Vent polymerase and 100 ng of genomic DNA. Thirtyfive cycles were run with denaturation at 95 °C for 30 s, annealing at 60 °C for 45 s and elongation at 72 °C for 1 min. For cloning of the PCR product, the following oligonucleotide primers flanking the PRNP open reading frame (ORF) were constructed to create the restriction enzyme recognition sequences NotI-MluI and KpnI-NheI, respectively (NotI and KpnI, underlined; MluI and NheI, underlined, bold italics): 5′-NotI-MluI, 5′-AAAAAGCGGCCGCACGCGTCCACCATGGCGAACCTTGGCTGC TGGATGCTG-3′ 3′-KpnI-NheI, 5′-AAAAAGGTACCGCTAGCTCATCCCACTATCAGGAAGATGAGG-3′ Cloning and Sequencing of the PCR Product. The PCR product was cleaved with NotI and KpnI restriction endonucleases and ligated into the pBluescript vector (Stratagene, Amsterdam, The Netherlands). The recombinant plasmid was transformed into competent DH5R Escherichia coli subsequently grown on LB agar with ampicillin (100 µg/mL). Colonies containing recombinant plasmids were selected and the inserts were sequenced using an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany). The complete sequence of the insert was determined. Subcloning and Plasmid Preparation. pBluescript containing PRNP-ORF was cleaved with MluI and NheI restriction endonucleases. Prior to ligation of the purified PRNP-ORF cleavage product into the bidirectional pBI-DsRed-EGFP Tet vector (Clontech, Palo Alto, CA), the vector was cleaved with MluI and NheI and the DsRed reporter gene of the vector was deleted. The resulting pBI-PRNP-EGFP construct was used for conditional expression in HEK 293 cells. pBi-PRNP-EGFP was then cut with MluI and XbaI (XbaI restriction site was located within NheI site) and the PRNP insert was subsequently ligated into CMV promotor containing mammalian expression vector pCMS-EGFP (Clontech, Palo Alto, CA) allowing for constitutive expression in Prnp0/0 cells. Plasmid DNA was prepared using the Qiagen Plasmid Maxi preparation kit (Qiagen, Heiden, Germany) HEK 293 Cell Line and Culture Conditions. HEK 293 cells (Stratagene, La Jolla, CA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich Chemie, Steinheim, Germany), supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), and 1% penicillin/streptomycin (PS) (Biochrom AG, Berlin, Germany) at 37 °C, 5% CO2 2682

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Ramljak et al. supply and 95% humidity. The cells were transiently transfected using calcium phosphate-mediated transfection procedure. Briefly, cells were seeded in 6-well plates at the cell density of 7 × 105 per well in DMEM supplemented with 10% FBS without trace levels of tetracycline (Tc) (BD Biosciences, Clontech, Palo Alto, CA). Use of this serum ensures the maximal range of induction with the Tet-Off system. In this system, gene expression is turned on in the absence of Tc or doxycycline (Dox; a Tc derivative) and turned off in the presence of Dox. The cells were ready for transfection after reaching approximately 50% confluence. Next, 437.5 µL of 2× HBS (Hepes buffered saline) was added to an equal volume of the mixture containing 15 µg of vector DNA, either pBI-PRNP-EGFP or control (pBI-DsRedEGFP), 3 µg of regulator plasmid (pTet-Off), CaCl2 and sterile water. The transfection mixture was briefly vortexed, spun down and incubated for 50 s at 25 °C in the water bath. Afterward, the mixture was immediately transferred into 7.9 mL of prewarmed DMEM containing 2% FBS without Dox, thoroughly mixed and distributed into the wells (2.5 mL/well). To suppress the gene expression in both pBI-PRNP-EGFP transfected and control transfected cells, Dox (1 µg/mL) was added into the culture medium. After an incubation period of 7-8 h, the transfection medium was replaced with the medium containing 10% FBS without Dox. Cells were collected from the confluent cultures 48 h after transfection. Transfection of cells with either pBI-PRNP-EGFP vector in the absence (PrP-Dox) and the presence of Dox (PrP+Dox) or with the control vector in the absence (ctrl-Dox) and the presence of Dox (ctrl+Dox) allowed for four different experimental conditions. PrPc Subcellular Localization Study. To assess the subcellular localization of PrPc, HEK 293 cells transfected with pBIPRNP-EGFP vector were grown on coverslips for either 24 or 48 h. Subsequently, the coverslips were collected from 8-well plates and washed in a phosphate-buffered saline (PBS). Cells were fixed by incubating the coverslips for 30 min with PLP (4% paraformaldehyde in PBS containing 1.2% L-lysine and 0.2% sodium-meta-periodate). After fixation, cells were permeabilized with 0.2% Triton X-100 in PBS, followed by 20 min blocking step using 0.2% casein-solution containing Tween 20. PrPc detection was carried out by applying the monoclonal mouse antibody 3F4 (1:200 in PBS, provided by M. Beekes, RKI, Berlin, Germany) for 90 min. The monoclonal antibody was detected by incubating slides for 60 min with a Cy3-labeled goat anti-mouse secondary antibody (1:200, Dianova, Hamburg, Germany). Incubation with Hoechst 33342 for 10 min was performed to visualize nuclei. Finally, coverslips were placed on glass slides and mounted with Fluoromount (DAKO, Hamburg, Germany). All the steps were carried out in a dark humid chamber and were stopped by washing coverslips three times with PBS. Control cells (expressing only endogenous PrPc) were harvested and processed under the same conditions as PrPcoverexpressing cells. Examination of the slides was performed on an Olympus BX51 microscope employing a fluorescence unit. Images were acquired and processed using cell F-software (Olympus). Prnp0/0 Cell Line and Culture Conditions. Cells were maintained in 60% DMEM, 30% Coon’s F12 modified medium (Biochrom AG, Berlin, Germany) with 10% FBS and 1% PS at 37 °C in a humidified atmosphere with 5% CO2. Transfection assays were performed using Lipofectamine 2000 (Invitrogen, Groningen, The Netherlands) following the supplier’s recommendations. Cells were collected from the confluent cultures 48 h after transfection. Transfection efficiency was estimated

Physiological Role of the Cellular Prion Protein (PrPc)

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to be between 6 and 15% by using FACS analysis (FACS Vantage, Becton Dickinson, Germany) Immunocytochemical Analysis of Prnp0/0 Cell Line. An SV40 immortalized Prnp0/0 murine brain cell line derived from Prnp0/0 transgenic mice21 was employed. To identify the expression of cell type specific markers, we performed immunostaining using the following antibodies: anti-synaptophysin (rabbit polyclonal, 1:50, DAKO, Hamburg, Germany), antineurofilament (NF) -200 kDa (mouse monoclonal, 1:100, Sigma, Taufkirchen, Germany), anti-nestin (mouse monoclonal, 1:200, Chemicon, Hofheim, Germany), anti-glial fibrillary acidic protein (GFAP) (rabbit polyclonal, 1:1000, DAKO) and antivimentin (mouse monoclonal, 1:300, DAKO). Cells were plated onto chamber slides (Lab-Tek Chamber Slide System, Nalge Nunc, Naperville, IL) at the cell density of (1-2) × 104 cells/ slide. After 48 h, cells were washed three times with Trisbuffered saline (TBS), fixed for 15 min in 100% ethanol or 4% paraformaldehyde, rinsed again with TBS and permeabilized for 15 min with 0.2% Triton X-100. Permeabilization was followed by blocking in TBS containing 0.1% Tween 20 and 0.2% casein for 10 min. Subsequently, the specimens were incubated with primary antibodies for 90 min at room temperature (RT), rinsed and incubated with the respective secondary antibodies. Secondary antibodies used were alkaline phosphatase (AP) conjugated goat anti-mouse (1:500, DAKO), AP-conjugated goat anti-rabbit (1:50, DAKO) and biotinylated sheep anti-mouse (1:200, Amersham Biosciences, Freiburg, Germany). Staining was performed after 20 min incubation with the freshly prepared substrate for AP detection which consisted of 0.01% (w/v) new fuchsin (Sigma, Taufkirchen, Germany), 0.02% sodium nitrite (Merck, Darmstadt, Germany), 0.028% naphtol-ASBi-phosphate (Sigma), 0.6% N,N-dimethylformamide (Sigma) in 0.05 M Tris-HCl buffer (pH 8.7) containing 1 mM levamisole (Sigma). Finally, the slides were lightly counterstained with hematoxylin solution, washed with water, and then mounted. The preparations were analyzed by light microscopy (Olympus BH2, Hamburg, Germany).

the proteins on a 7 cm IPG strip was initiated at 200 V for 2 h, followed by ramping at 500 V for 2 h, and final focusing at 4000 V for 5 h for a total of 20 000 Vh. The 17 cm IPG strips were focused at 500 V for 1 h; 1000 V for 1 h and finally at 8000 V, reaching the total of 32 000 Vh at the end. After IEF separation, proteins immobilized on the IPG strip were reduced in the buffer containing 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol, 2% DTT, and 0.375 M Tris-HCl (pH 8.8) for 25 min and were alkylated in the same buffer supplemented with 2.5% iodoacetamide instead of DTT for a further 25 min. Equilibrated strips were placed on top of vertical 12% polyacrylamide gels and electrophoresis was carried out either at 100 V for 2 h (Mini Protean II gel chamber) or at 100 V overnight (Protean xi 2-D Cell) at 4 °C. After electrophoresis, 2-DE gels were silver-stained,22 Coomassie-stained (Roth, Karlsruhe, Germany) or were immunoblotted as described in Western Blotting Section. Gels were scanned using ScanMaker 4 (Microtek, International) and densitometric analyses were carried out using Delta 2D (Decodon, Greifswald, Germany) software. For each condition analyzed, three gels were prepared from three different protein extractions. Differences in spot abundance detected by densitometric software were statistically evaluated using unpaired Student’s t test. Means and standard deviations were calculated from three independent sets of experiments. The differences in protein expression with p-value < 0.05 were considered significant. Identification of Protein/Peptide Sequence Analysis. In-gel digestion was carried out according to a modified published protocol.23 Spots of interest were excised from the silver-stained gel into 1-2 mm2 slices, destained with 15 mM potassium ferricyanide/50 mM sodium thiosulfate (Aldrich/Sigma-Aldrich, Steinheim, Germany) and then equilibrated with 50 mM ammonium bicarbonate/50% acetonitrile (ACN) (Sigma-Aldrich). Samples were dried for 15 min using the SpeedVac SVC100 (Savant Instruments, Farmingdale, NY) vacuum concentrator. The dried spots were rehydrated on ice with 10-20 µL of trypsin digestion solution (Promega, Madison, WI) for 45 min followed by an overnight incubation at 37 °C in digestion solution without trypsin. The peptides were first extracted with 0.1% trifluoracetic acid (TFA) for 30 min in the sonicating water bath Transsonic 310/H (Elma, Pforzheim, Germany) followed by extraction with 30% ACN in 0.1% TFA and 60% ACN in 0.1% TFA. The eluate was collected in Eppendorf tubes and dried with the SpeedVac. The extracted peptides were dissolved in 0.1% formic acid (FA) for mass spectrometric analysis as described earlier.24

Two-Dimensional Gel Electrophoresis (2-DE) and Image Analysis. HEK 293 cells were transfected with pBI-PRNP-EGFP, whereas Prnp0/0 cells were transfected with pCMS-PRNP-EGFP vector. Empty vector controls were used in parallel. Forty-eight hours post-transfection, the cells were washed with cold PBS, scraped and centrifuged at 4 °C, ∼4000g for 20 min. The supernatant was decanted and the pellet was resuspended in cold PBS and centrifuged again at 4 °C, ∼4000g for 10 min. Cells were lysed in 7 M urea, 2 M thiourea, 4% CHAPS, 2% Ampholytes, 1% dithiothreitol (DTT) and a protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride, 10 µM N-Rp-tosyl-L-lysine chloromethyl ketone and 10 µM L-1-tosylamide2-phenylethyl-chlorometyl ketone). The lysate was centrifuged in a microcentrifuge at 14 000 rpm for 10 min/4 °C to remove cell debris. Protein concentration was determined by the Bradford assay (Bio-Rad, Mu ¨nchen, Germany). Protein samples were diluted with rehydration buffer (7 M urea, 2 M thiourea, 15 mM DTT, 4% CHAPS, 2% Ampholytes) for first-dimension isoelectric focusing (IEF). The amount of protein loaded on each immobilized pH gradient (IPG) strip (Bio-Rad, Mu ¨ nchen, Germany) varied with the staining method and the length of the strip. IEF on a 7 cm IPG strip (pH 3-10, linear) was performed by applying 40 µg of proteins per strip (both cell lines). The protein load on a 17 cm IPG strip (pH 3-10, linear) for Coomassie staining was 400 µg (HEK 293 cells), whereas for silver staining, it was 130 µg (Prnp0/0 cells). The focusing of

One microliter of each sample was introduced using a CapLC auto sampler (Waters) onto a µ-precolumn cartridge C18 pepMap (300 µm × 5 mm; 5 µm partical size) and further separated through a C18 pepMap100 nano Series (75 µm ×15 cm; 3 µm partical size) analytical column (LC Packings). The mobile phase consisted of solution A (0.1% FA in 5% ACN) and solution B (0.1% FA in 95% ACN). The single sample run time was set for 60 min. The chromatographically separated peptides were then analyzed on a Q-TOF Ultima Global (Micromass, Manchester, U.K.) mass spectrometer equipped with a nanoflow ESI Z-spray source in positive ion mode. The data acquisition was performed using MassLynx (v 4.0) software on a Windows NT PC and data were further processed on ProteinLynx-Global-Server (v 2.1), (Micromass, Manchester, U.K.). Processed data were searched against MSDB and Swiss-Prot databases through the Mascot search engine using a peptide mass tolerance of 100 ppm and fragment tolerance of 0.5 Da. Journal of Proteome Research • Vol. 7, No. 7, 2008 2683

research articles The search criteria were set with one missed cleavage by trypsin allowed and protein modifications set to methionine oxidation and carbamidomethylcysteine when appropriate. Western Blotting. To perform 2-DE Western blots, the cell pellets were resuspended in the lysis buffer described in TwoDimensional Gel Electrophoresis (2-DE) and Image Analysis Section. To perform one-dimensional Western blots, the pellets were resuspended in a lysis buffer containing 0.5% SDS, 50 mM Tris-HCl, pH 8.0, and 1 mM DTT. Equal amounts of proteins were separated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF) (AppliChem, Darmstadt, Germany). The PVDF membranes were blocked with 5% (v/v) nonfat dry milk in PBS and 0.1% Tween 20 (PBST) for 1 h at RT. Subsequently, the membranes were incubated with the primary antibodies antiPrP 12F10 monoclonal antibody25 (1:1000), anti-annexin V polyclonal antibody (Abcam, Cambridge, U.K.; 1:250), antiannexin II monoclonal antibody (BD Transduction Laboratories, Palo Alto, CA; 1:5000), anti-lacate dehydrogenase polyclonal antibody (Abcam, 1:500) or anti-β actin monoclonal antibody (Abcam, 1:5000) overnight at 4 °C. Membranes were then rinsed in PBST and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (diluted 1:2000/1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at RT. Immunoreactivity was detected after immersion of the membranes into enhanced chemiluminescence (ECL) solution and exposition to ECL-Hyperfilm (Amersham Biosciences, Buckinghamshire, U.K.). Densitometric values for each sample were obtained using the ScanMaker4 (Microtek, International), after correction for the background. ELISA. ELISA (enzyme-linked immunosorbent assay) was performed using commercially available BetaPrion BSE EIA Test Kit according to supplier’s recommendations (Leipzig, Germany).

Results HEK 293 Cell Line. Conditional Tet-Off expression system was employed to investigate protein expression changes induced by different levels of PrPc expression in HEK 293 cells. HEK 293 cells were transiently transfected with PRNP containing vector either in the absence (PrP-Dox) or the presence of Dox (PrP+Dox). The Tet-Off system provided high expression of PRNP gene in the induced state (PrP-Dox) and moderate expression in the noninduced state (PrP+Dox). The expression of coregulated reporter gene, enhanced green fluorescence protein (EGFP), enabled estimation of the level of PrPc expression under both experimental conditions (Figure 1D,E). The fluorescence intensity of EGFP in HEK 293 cells transfected with pBI-PRNP-EGFP vector in the absence of Dox (Figure 1D) was markedly higher than the fluorescence intensity in the presence of 1 µg/mL Dox (Figure 1E). The corresponding controls, HEK 293 cells transfected with the control vector in the absence (ctrl-Dox) or the presence of Dox (ctrl+Dox), were used in parallel (Figure 1A-C). Western blot analysis showed that PrPc expression level in HEK 293 cells overexpressing PrPc in the absence of Dox was notably higher than PrPc level observed in the presence of Dox (Figure 1F). The control cells, expressing only the endogenous PrPc, exhibited markedly lower PrPc expression levels as compared to PrPc-overexpressing cells, under both experimental conditions (Figure 1F). Thus, the data obtained by Western blot analysis matched well with those of fluorescence microscopy presented in Figure 1A-E. In addition, we used an ELISA test to quantify PrPc levels in the cell lysates, 2684


Ramljak et al. subsequently used for 2-DE analyses. The mean values of PrPc level in the cells overexpressing PrPc, either in the absence (75.4 ( 12.6 ng/mL) or in the presence of Dox (44.7 ( 1.7), were significantly different (p < 0.05). The endogenous level of PrPc expression in the control groups was under the detection limit of ELISA. The proteomes of four distinct groups including ctrl-Dox, ctrl+Dox, PrP-Dox and PrP+Dox were compared in order to reveal the changes in protein expression profiles. Five pairwise proteome comparisons were carried out. Thus, the ctrl-Dox group (a reference group) was compared to ctrl+Dox, PrP-Dox and PrP+Dox groups. In addition, PrP+Dox and ctrl+Dox groups were compared with each other due to the presence of Dox in the culture medium and PrP-Dox with PrP+Dox groups due to different levels of PrPc overexpression. Densitometric analysis of Coomassie-stained 2-DE gels revealed a total of 36 differentially regulated protein spots (representing 28 proteins) (Table 1.). Observed individual ion scores showing the homology between sequenced peptides and corresponding proteins that were higher than 25 (p < 0.05) indicated identity or extensive homology. The threshold for identification of upregulated/down-regulated proteins was set to at least 2-fold change. All the proteins listed in Table 1 exhibited significant differences in their regulation as calculated by an unpaired Student’s t test. The comparison of the protein profiles between different groups revealed the following: nine significantly regulated proteins between ctrl-Dox and ctrl+Dox; six between ctrl-Dox and PrP-Dox; 13 between ctrl-Dox and PrP+Dox; two between PrP-Dox and PrP +Dox and five between ctrl+Dox and PrP+Dox group. The locations of differentially regulated spots after transfection of HEK 293 cells are shown in Figure 2A. All the proteins that were found to be regulated by PrPc, following the comparison of ctrl-Dox/PrP-Dox and ctrl-Dox/ PrP+Dox group, but at the same time exhibited differential regulation between both control groups, ctrl-Dox/ctrl+Dox, were considered not to be regulated by PrPc (see accession numbers highlighted by asterisks in Table 1.) and omitted from further discussion. Our presumption was that the regulation of proteins observed between the two control groups was very likely the consequence of transfection procedure and/or presence of Dox. An interesting side finding of our study was Dox induction of elfin (Figure 2A spot no. 113) mobility shift. This is fully in line with the aforementioned assumption that Dox itself might affect protein expression pattern of HEK 293 cells. The presence of Dox in the culture medium of cells treated either with PRNPcontaining or control vector led to a marked decrease in molecular weight of elfin, a cytoskeletal protein, as compared to the corresponding matches cultured in the absence of Dox [Supporting Information Figure 8]. 1. Identification of Differentially Expressed Proteins between Ctrl-Dox/PrP-Dox Groups. The differences in protein profiles between HEK 293 cells expressing low (ctrl-Dox) and high level of PrPc (PrP-Dox) revealed specific, prominent regulation of five proteins (Table 1). Four proteins found to be significantly down-regulated by high level of PrPc expression are involved in energy metabolism: 3-hydroxyacyl-CoA dehydrogenase type II (HCD2) (Figure 2B), NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 and electron transfer flavoprotein subunit beta. One protein is related to calcium, calcyclin-binding protein. The only protein up-regulated by PrPc, splicing factor U2AF35 kDa subunit, is implicated in RNA metabolism.


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Figure 1. Expression of reporter genes, and Western blot analysis of PrPc expression, following transient transfection of HEK 293 cells using the Tet-Off inducible system. Both reporter genes, EGFP (A) and DsRed (B), displayed high fluorescence intensity in pBI-DsRedEGFP (control) transfected cells in the absence of Dox whereas 1 µg/mL of Dox in culture medium resulted in suppressed EGFP (C) and DsRed expression (data not shown). Likewise, induced and reduced EGFP expression was seen in pBI-PRNP-EGFP transfected cells in the absence and the presence of 1 µg/mL Dox, respectively (D and E). Scale bar in D (100 µm) is applicable to panels A-E. In panel F, all lanes (1-4) were loaded with 10 µg of proteins from total cell lysate. Lanes 1 and 3 were loaded with the lysate prepared from pBI-PRNP-EGFP transfected cells in the absence and the presence of 1 µg/mL Dox, respectively. PrPc expression level in the absence of Dox (lane 1) is markedly higher than in the presence of Dox (lane 3), confirming a fine regulation of the Tet-Off system. Basal, faintly visible, levels of endogenous PrPc expression were obtained from the lysates prepared from the control transfected cells, either in the absence (lane 2) or in the presence of 1 µg/mL Dox (lane 4). The Western blot is representative of three independent experiments.

2. Identification of Differentially Expressed Proteins between Ctrl-Dox/PrP+Dox Groups. Among 10 specifically regulated proteins detected in HEK 293 cells expressing low level of PrPc (ctrl-Dox) versus cells exhibiting moderate level of PrPc expression (PrP+Dox), five proteins were down-regulated and five up-regulated by PrPc. Interestingly, 3-hydroxyacyl-CoA dehydrogenase type II, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 and calcyclin-binding protein already shown to be down-regulated by PrP-Dox exhibited several fold down-regulation by PrP+Dox as well (Table 1). Another two down-regulated proteins are stress-related chaperone, 78 kDa glucose-regulated protein precursor (GRP78) and NEDD8 conjugating enzyme Ubc 12 playing a role in neddylation. Five remaining up-regulated proteins are related to oxidative stress, glutathione S-transferase P, glucose metabolism, alpha enolase, protein degradation, COP9 signalosome complex subunit 4 and DNA replication/chromosome segregation, DNA replication licensing factor MCM3 and glutamate rich WD repeat-containing protein 1.

3. Identification of Differentially Expressed Proteins between PrP-Dox/PrP+Dox Groups. The comparison of 2D maps between high (PrP-Dox) and moderate level of PrPc expression (PrP+Dox) revealed only two significantly different regulated proteins (Table 1). Eukaryotic translation initiation factor 4H was up-regulated by PrP-Dox, while mitotic checkpoint protein BUB3 (BUB3) (Figure 2B), important for cell cycle progression, was up-regulated by PrP+Dox. 4. Identification of Differentially Expressed Proteins between Ctrl+Dox/PrP+Dox Groups. The differences in protein expression profiles between HEK 293 cells expressing low, endogenous level of PrPc in the presence of Dox (ctrl+Dox) and the cells exhibiting moderate level of PrPc expression in the presence of Dox (PrP+Dox) consisted of five proteins. Four out of five proteins showed a significant increase in expression following transfection by PrPc, while one protein showed a significant decrease (Table 1). Up-regulated proteins included a cochaperone, activator of 90 kDa heat shock protein ATPase homologue 1, ATP synthase subunit alpha (ATPA) involved in Journal of Proteome Research • Vol. 7, No. 7, 2008 2685

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Table 1. List of Proteins Identified from 2-DE Gels of HEK 293 Cells groups compared

ctrl-Dox/ctrl+Dox

ctrl-Dox/PrP-Dox

ctrl-Dox/PrP+Dox

PrP-Dox/PrP+Dox

+Dox

PrP

+Dox

/ctrl

spot no.

fold change

20 22 57

a

peptides matched

score

state change

P value

0.49 0.44

13 12

139 250

vctrl+Dox vctrl+Dox

0.02 0.04

0.50

19

417

vctrl+Dox

0.003

+Dox

66

0.38

8

140

vctrl

0.03

127 156

7.03 2.42

8 14

284 297

ctrl-Dox Vctrl+Dox

0.01 0.007

163 224 206

2.00 2.09 2.00

2 22 3

51 441 108

ctrl-Dox ctrl-Dox ctrl-Dox

0.04 0.03 0.03

116

0.50

1

37

vPrP-Dox

0.05

-Dox

127 128

10.55 3.47

8 8

284 184

VPrP VPrP-Dox

0.01 0.0003

185 186

2.44 2.25

5 20

110 564

VPrP-Dox VPrP-Dox

0.001 0.05

201

2.54

6

194

VPrP-Dox

0.006

13

0.47

7

240

vPrP+Dox

0.001

22

0.34

12

250

vPrP+Dox

0.003

+Dox

32

2.25

21

862

VPrP

0.009

90

0.43

11

197

vPrP+Dox

0.02

+Dox

127 128

8.68 3.31

8 8

284 184

VPrP VPrP+Dox

0.01 0.0005

156

2.07

14

297

VPrP+Dox

0.009

+Dox

159

2.05

6

313

VPrP

0.03

185 201

2.31 3.70

5 6

110 194

VPrP+Dox VPrP+Dox

0.004 0.004

208 258

0.44 0.43

17 6

430 54

vPrP+Dox vPrP+Dox

0.001 0.05

286 136

0.48 2.51

12 2

232 70

vPrP+Dox vPrP-Dox

0.01 0.05

250

0.50

5

154

vPrP+Dox

0.0001

+Dox

70 76 87

2.30 2.41 2.76

7 8 3

198 201 49

vPrP vPrP+Dox vPrP+Dox

0.04 0.03 0.03

119 159

2.57 0.44

3 6

78 313

vPrP+Dox VPrP+Dox

0.04 0.03

2

46

113

post-translational

protein ID

heat shock protein 90 beta ATP dependent DNA helicase 2 subunit 2 pyruvate kinase isozymes M1/M2 spliceosome RNA helicase BAT1 adenylate kinase 2 nucleoside diphosphate kinase A prefoldin subunit 2 elongation factor 2 proteasome subunit beta type 3 splicing factor U2AF 35 kDa subunit adenylate kinase 2 3-hydroxyacyl-CoA dehydrogenase type II calcyclin-binding protein electron transfer flavoprotein subunit beta NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 DNA replication licensing factor MCM3 ATP dependent DNA helicase 2 subunit 2 78 kDa glucose-regulated protein precursor COP9 signalosome complex subunit 4 adenylate kinase 2 3-hydroxyacyl-CoA dehydrogenase type II nucleoside diphosphate kinase A NEDD8 conjugating enzyme Ubc 12 calcyclin-binding prot. NADH dehydrogenase (ubiquinone) iron- sulfur protein 3 glutathione S-transferase P glutamate rich WD repeat-containing protein 1 alpha-enolase eukaryotic translation initiation factor 4H mitotic checkpoint protein BUB3 ATP synthase subunit alpha elongation factor 1-alpha 1 activator of 90 kDa heat shock protein ATPase homologue 1 annexin II NEDD8 conjugating enzyme Ubc 12 PDZ and LIM domain protein 1

accession no.

P08238 P13010* P14618 Q13838 P54819* P15531* Q9UHV9 P13639 P49720 Q01081 P54819* Q99714 Q9HB71 P38117 O75489

P25205 P13010* P11021 Q9BT78 P54819* Q99714 P15531* P61081 Q9HB71 O75489

P09211 Q9BQ67 P06733 Q15056 O43684 P25705 P68104 O95433

P07355 P61081 O00151

a Twenty-eight different proteins were identified from 2-DE gels of HEK 293 cells transiently transfected either with pBI-PRNP-EGFP or control vector in the absence/presence of 1 µg/mL Dox. The number of the spots corresponds to their location on the gel (Figure 2A). The comparison of protein regulation between distinct transfection groups, fold change, number of peptides matched, ion score, state change, significance (unpaired Student’s t test), protein identification and Swiss-Prot accession numbers have been given for each spot except for the spot number 113. This protein, although not regulated by PrPc, is shown on the gel due to its mobility shift in the presence of Dox. The depiction of transfection groups is as follows: ctrl-Dox ) pBI-DsRed-EGFP Dox; ctrl+Dox ) pBI-DsRed-EGFP + Dox; PrP-Dox ) pBI-PRNP-EGFP - Dox; and PrP+Dox ) pBI-PRNP-EGFP + Dox. The proteins marked by asterisks were not regulated by PrPc (see in the text).

energy production (Figure 2B), annexin II, a calcium binding protein, and elongation factor 1-alpha 1 implicated in cytoskeletal rearrangements. Given annexin II down-regulation fol2686


lowing an introduction of PRNP into Prnp0/0 cells (Figure 6), annexin II up-regulation by PrPc overexpression in HEK 293 cells was further examined. Densitometric analysis of 2-DE


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Figure 2. Coomassie-stained 2-DE gel of HEK 293 cells transiently transfected with pBI-PRNP-EGFP vector in the absence of Dox. Linear 17 cm IPG strips (pH 3-10) were used and loaded with 400 µg of proteins. Labelling on the gel represents the location of 28 protein spots detected as differentially regulated between distinct transfection groups (A). The protein identity of spots is listed in Table 1. The location of the spot number 113, although not regulated by PrPc, was included due to its regulation by Dox (see in the text). Panel B shows PrPc-induced regulation of three proteins, HCD2, BUB3 and ATPA regulated between distinct transfection groups. The horizontal axes represent compared transfection groups, while the vertical axes stand for relative regulation of protein expression in terms of fold change. HCD2 (spot number 128), a protein associated with AD and PD, was markedly down-regulated by PrPc overexpression in the absence of Dox as compared to appropriate control. BUB3 (spot number 250), a protein involved in cell cycle control, showed highly significant regulation between different levels of PrPc overexpression. ATPA (spot number 70), a protein involved in energy metabolism, was significantly up-regulated by PrPc overexpression in the presence of 1 µg/mL Dox compared with the corresponding control. Means and standard deviations were calculated from three independent sets of experiments using an unpaired Student’s t test. Levels of significance: *, p < 0.05; ***, p < 0.001.

Western blot additionally confirmed 2.6-fold up-regulation of Anxa II by PrPc (Figure 3) as already shown by densitometric analysis of Coomassie-stained gels (Table 1). The only protein down-regulated by PrP+Dox is NEDD8 conjugating enzyme Ubc 12. The decreased expression of the latter protein by PrP+Dox was already detected after the comparison of ctrl-Dox versus PrP+Dox 2D maps. 5. PrPc Subcellular Localization. HEK 293 cells successfully transfected with PRNP were identified by their green, EGFP fluorescence (Figure 4A,C, D,F) Twenty-four hours after transfection, EGFP-positive cells displayed specific staining for PrPc (Figure 4B,C) predominantly located in vesicular structures next to the nucleus, and to a very low extent at the cell surface. After 48 h, PrPc was mainly detected at the cell surface (Figure 4E,F). We interpret this result as an indication for the translocation of PrPc from the Golgi apparatus and rough endoplasmic reticulum to the cell membrane. Prnp0/0 Cell Line. To immunocharacterize the Prnp0/0 murine brain cell line, we performed staining using different cell type specific markers. Control staining with specific secondary antibodies was done in parallel. Immunostaining with anti-rat-nestin antibody resulted in moderate reaction suggesting that the cells employed in this study belong to

Figure 3. 2-DE Western blot analysis showing Anxa II upregulation following PrPc overexpression in HEK 293 cells in the presence of Dox. (A) Regulation of Anxa II (38 kDa, pI ) 7.6) in HEK 293 cells transfected with the control vector in the presence of 1 µg/mL Dox (horizontal arrow). (B) Regulation of Anxa II in HEK 293 cells transfected with pBi-PRNP-EGFP vector in the presence of 1 µg/mL Dox. Note a prominent upregulation of Anxa II by overexpression of PrPc as compared to the control. An equal protein load (40 µg) of linear IPG strips (pH 3-10) is shown by beta-actin expression (vertical arrow). The displayed Western blot is representative of three independent experiments. Journal of Proteome Research • Vol. 7, No. 7, 2008 2687

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Figure 4. Subcellular distribution of PrPc in PrPc-overexpressing HEK 293 cells. Twenty-four hours post-transfection (A-C) PrPc (red fluorescence) is located predominantly next to the nucleus (blue fluorescence) of transfected (green fluorescence) cells. Forty-eight hours post-transfection (D-F) PrPc is redistributed at the cell surface.

neuroepithelium and are not fully differentiated. Staining with anti-vimentin and anti-GFAP antibody, characteristic for the cells of mesenchymal and glial origin, was negative, while the antibodies specific for the cells of neuronal origin, antisynaptophysin and anti-NF-200 kDa gave strong and weak reaction, respectively (Supporting Information Figure 9). The results suggest that Prnp0/0 cells used in this study are neuronal progenitor cells. PrPc expression level after transient transfection of Prnp0/0 cells with PRNP-containing vector was very low (Figure 5B). Because of low transfection efficiency (6-15%), we decided to first use 7 cm IPG strips to examine whether such a low amount of PrPc can already induce detectable proteome changes. Densitometric evaluation of silver-stained 2-DE gels revealed two protein spots exhibiting prominent expression changes between PRNP-containing and the control vector transfected Prnp0/0 cells (Figure 5A). The first spot was identified as annexin V (Anxa V), while the second one was found to be a combination of two unresolved proteins, Anxa II and lactate dehydrogenase A (LDH-A). A total of 19 peptides were sequenced and subsequently matched to Anxa V (accession 2688


number P48036) with the high score of 651. Seventeen peptides were matched to Anxa II (accession number P07356) and 4 to LDH-A (accession number P06151) both with highly significant scores of 832 and 223 as compared to the protein sequence coverage in the MS/MS analysis. Our results obtained on silverstained 2-DE gels were confirmed by 2-DE Western blots using antibodies specific for Anxa V (Figure 6 panels A1, A2), Anxa II (Figure 6 panels B1, B2) and LDH-A (Figure 6 panels C1, C2). Beta-actin antibody was used as a control for an equal protein load (Figure 6 panels D1, D2). Densitometric quantification of three different Western blots revealed significant (p < 0.05) 1.5fold up-regulation of Anxa V, 1.3-fold down-regulation of Anxa II, and 2-fold up-regulation of LDH-A in pCMS-PRNP-EGFP transfected Prnp0/0 cells as compared to the controls. Remarkably, Anxa II was already shown to be markedly up-regulated by PrPc-overexpressing HEK 293 cells in the presence of Dox. To expand the proteome map of Prnp0/0 cells, we performed 2-DE analysis using 17 cm IPG strips. To avoid losing relevant information due to low transfection efficiency, we adjusted the threshold of fold change for identification of up- and down-


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Figure 5. 2-DE gel differential protein expression patterns of pCMS-PRNP-EGFP and control vector transfected Prnp0/0 cells. (A) Linear 7 cm silver-stained 2-DE-gel (pH 3-10) of pCMS-EGFP transfected Prnp0/0 cells. The insets illustrate magnified parts of 2-DE gels from pCMS-EGFP (panels A1, B1, C1) and pCMS-PRNP-EGFP (panels A2, B2, C2) transfected Prnp0/0 cells. Panels A1, A2 show differential protein expression levels of Anxa V (36 kDa, pI ) 4.8), while panels B1, B2 show Anxa II/LDH-A (38 kDa, pI ) 7.6/ 36 kDa, pI ) 7.6) expression. Expression levels of a nonregulated protein spot (disulfide-isomerase A3 precursor) are displayed in panels C1, C2. (B) Detection of PrPc by Western blot in Prnp0/0 cells transiently transfected with pCMS-PRNP-EGFP vector. The detection of PrPc in 10 µg of proteins prepared from the total cell lysate of pCMS-PRNP-EGFP transfected Prnp0/0 cells. PrPc was not detectable in the cell lysate of control transfected Prnp0/0 cells (data not shown).

regulated proteins to 1.5. The differences between the proteome maps of Prnp0/0 cells transfected with PRNP -containing vector versus control revealed a further 11 significantly regulated protein spots (Table 2). Three protein spots, 253, 427 and 605, remained unidentified. The locations of all significantly regulated proteins, as calculated by an unpaired Student’s t test, are shown in Figure 7A. Six out of eleven proteins were upregulated including superoxide dismutase (SODC) (Figure 7B), elongation factor 1-alpha 1, heat shock protein 27 (Hsp27), peptidyl-tRNA hydrolase 2, and two unidentified protein spots, 253 and 605, while five were down-regulated: heterogeneous nuclear ribonucleoproteins C1/C2 (HNRPC) (Figure 7B), transaldolase (TALDO) (Figure 7B), eIF3 beta TGF beta receptor interacting protein, prolyl 4-hydroxylase alpha-1 subunit precursor and unidentified 427 protein spot. A high proportion of the proteins regulated after the introduction of PRNP into Prnp0/0 cells were related to protection against oxidative stress and thus prevention of cell death.

Discussion HEK 293 Cell Line. Several lines of evidence demonstrated that overexpression of PrPc can exert a detrimental effect both in vitro and in vivo.14,15,26 Lately, it was reported that an overexpression of N-terminally truncated PrPc proteolytic fragment (C1) in two different cell lines leads to an increased sensitivity to apoptotic stimuli through modulation of p53.27 Moreover, PrPc-induced myopathy in transgenic mice overexpressing wild-type PrPc positively correlated with accumulation of C1 fragment in skeletal muscle.15 It appears that the damaging effects of PrPc overexpression are the consequence of a higher production of C1 fragment. However, the proteins modulated by PrPc overexpression, causing the observed proapoptotic phenotypes, are largely unknown. To identify pro-

teins associated with PrPc overexpression, five pairwise proteome comparisons between distinct transfection groups, expressing different levels of PrPc, were carried out. The comparison between the two control groups (ctrl-Dox/ctrl+Dox), used to distinguish between PrPc-regulated and not regulated proteins, was omitted from further discussion. 1. Differentially Expressed Proteins between Ctrl-Dox/ PrP-Dox Groups. The comparison between endogenous, low level of PrPc expression (ctrl-Dox) and a high PrPc expression (PrP-Dox) revealed prominent regulation of the following proteins: 3-hydroxyacyl-CoA dehydrogenase type II, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, electron transfer flavoprotein subunit beta, calcyclin-binding protein and splicing factor U2AF35 kDa subunit. The expression of 3-hydroxyacyl-CoA dehydrogenase type II, better known as endoplasmic reticulum amyloid-β binding protein (ERAB), a predominantly mitochondrial enzyme was more than 3-fold decreased in PrP-Dox group as compared to the control group. ERAB was shown to be increased in AD affected neurons and to enhance Aβ cytotoxicity in neuroblastoma and COS-1 cells.28 Conversely, another study suggested a positive correlation between low levels of ERAB and deposition of amyloid-β by brain smooth muscle cells.29 Furthermore, ERAB was shown to be down-regulated in substantia nigra pars compacta of Parkinson disease (PD) patients and its overexpression reduced dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) mouse model.30 Given ERAB’s regulation by high level of PrPc expression in the present culture model, and the previously shown association with AD and PD, further studies are required to address the question of possible ERAB role in the pathogenesis of CreutzfeldtJakob disease (CJD). Two additional proteins essential for cellular energy metabolism, NADH dehydrogenase (ubiquinoJournal of Proteome Research • Vol. 7, No. 7, 2008 2689

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Figure 6. 2-DE Western blot comparison of Anxa V, Anxa II and LDH-A synthesis between pCMS-PRNP-EGFP vector transfected and control vector transfected Prnp0/0 cells. Linear 7 cm IPG strips (pH 3-10) were used and loaded with equivalent protein amounts (40 µg) as shown by beta-actin expression (panels D1, D2). Panels A1, B1 and C1 represent pCMS-PRNP-EGFP transfected Prnp0/0 cells, while panels A2, B2 and C2 represent control vector transfected Prnp0/0 cells. The expression levels of Anxa V (panels A1, A2) and LDH-A (panels C1, C2) were increased in pCMS-PRNP-EGFP transfected Prnp0/0 cells, whereas Anxa II expression (panels B1, B2) was decreased. The displayed Western blots are representatives of three different experiments, each (p < 0.05). Table 2. List of Proteins Identified from 2-DE Gels of Prnp0/0 Cellsa groups compared

PrP+/+/PrP0/0

spot no.

fold change

peptides matched

score

state change

P value

protein ID

accession no.

108 143 214 253 274 355 408 427 436 592 605

1.59 0.27 1.78 1.61 2.03 7.41 0.44 0.41 0.48 0.44 1.70

2 2 5 / 3 3 2 / 2 2 /

33 40 132 / 76 132 28 / 56 73 /

vPrP+/+ VPrP+/+ vPrP+/+ vPrP+/+ vPrP+/+ vPrP+/+ VPrP+/+ VPrP+/+ VPrP+/+ VPrP+/+ vPrP+/+

0.007 0.002 0.01 0.03 0.03 0.01 0.04 0.02 0.007 0.05 0.04

elongation factor 1-alpha 1 transaldolase heat shock 27 kDa protein / peptidyl t-RNA hydrolase 2 superoxide dismutase Cu-Zn eukaryotic translation initiation factor 3 subunit 2 / heterogeneous ribonucleoproteins C1/C2 prolyl 4-hydroxylase alpha-1 subunit precursor /

P10126 Q93092 P14602 / Q8R2Y8 P08228 Q9QZD9 / Q9Z204 Q60715 /

a Eleven proteins identified from 2-DE gels of Prnp0/0 cells transiently transfected either with pCMS-PRNP-EGFP or control vector with spot number corresponding to their location on the gel (Figure 7A). A fold change in protein regulation between two transfection groups, as well as number of peptides matched, ion score, state change, significance (an unpaired Student’s t test), protein identification and Swiss-Prot accession numbers have been given for each spot.

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Figure 7. Silver-stained 2-DE gel of Prnp0/0 cells transiently transfected with pCMS-PRNP-EGFP vector. Linear 17 cm IPG strips (pH 3-10) were used and loaded with 130 µg of proteins. Labelling on the gel represents the location of the relevant spots (A). The protein identity of the spots is listed in Table 2. Panel B shows PrPc-induced regulation of three proteins belonging to three different groups of proteins, involved in regulation of oxidative stress (SODC), RNA metabolism (HNRPC) and energy metabolism (TALDO). The horizontal axes represent different transfection groups, while the vertical axes represent relative regulation of protein expression as fold change. SODC (spot number 355) was several fold up-regulated by introduction of PrPc in Prnp0/0 cells, while HNRPC (spot number 436) and TALDO (spot number 143) were both markedly down-regulated. Means and standard deviations were calculated from three independent sets of experiments using an unpaired Student’s t test. Levels of significance: *, p < 0.05; **, p < 0.01.

ne) iron-sulfur protein 3 and electron transfer flavoprotein subunit beta, displayed more than 2-fold down-regulation by PrPc overexpression. The former protein belongs to the complex I 30 kDa subunit family. Complex I deficiency was reported in the brains of both PD and AD patients, suggesting an impaired cellular energy production.31,32 Recently, a study based on DNA microarray analysis, showed reduced expression of NADH ubiquinone oxidreductase chain 6 and chain 5 following treatment of SH-SY5Y neuroblastoma cells with neurotoxic prion peptide 106-126.18 Growing evidence points out that alterations in regulation of calcium and calcium-related proteins play an important role in neurodegeneration.33,34 The calcyclin-binding protein was shown to be highly abundant in brain tissue and to be involved in neuronal differentiation.35,36 Although its biological function is still unclear, a role in calcium signaling was proposed.35 Our finding that PrPc overproduction in HEK 293 cells diminishes its expression level is in accordance with a recent report demonstrating, by microarray analysis, a decreased expression of calcyclin-binding protein upon transfection of human gastric cancer cells with PrPc.37 The only protein showing an upregulation by PrPc overexpression after comparing the protein profiles of ctrl-Dox/PrP-Dox was splicing factor U2AF35 kDa subunit. Briefly, our results obtained from the comparison of protein profiles between PrPc-overexpressing HEK 293 cells in the

absence of Dox and appropriate controls unequivocally point to disturbed cellular homeostasis upon PrPc overexpression. Decreased regulation of proteins important for energy production upon PrPc overproduction might be the reason for the previously reported increased sensitivity of PrPc-overexpressing HEK 293 cells and other cell lines to apoptotic stimuli.38 The established link between PrPc-mediated regulation of ERAB and NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 and earlier reports on regulation of these proteins in AD and PD is suggestive of common molecular mechanisms underlying the pathogenesis of distinct neurodegenerative disorders. To show that the observed effects of PrPc overexpression are not the consequence of excess of PrPc accumulating in the cytosol without reaching the plasma membrane, we managed to show that in the time course of transfection PrPc indeed translocates from the vesicular structures (24 h post-transfection) to the plasma membrane (48 h post-transfection). 2. Differentially Expressed Proteins between Ctrl-Dox/ PrP+Dox Groups. Differences in protein profiles following the comparison between low level of PrPc expression (ctrl-Dox) and moderate level of PrPc expression (PrP+Dox) were found in expression of ERAB, calcyclin-binding protein and NADH dehydrogenase (ubiquinone) iron-sulfur protein 3. These three proteins, already detected as markedly down-regulated by high level of PrPc expression (PrP-Dox), exhibited the same trend in regulation by a moderate level of PrPc expression (PrP+Dox). Journal of Proteome Research • Vol. 7, No. 7, 2008 2691

research articles Moreover, these proteins did not show any differences in the expression between the control groups. This reinforcing finding emphasizes once again the importance that PrPc-mediated regulation of these three proteins might have for cell physiology. Other proteins whose regulation corroborated the picture of perturbed cellular equilibrium in HEK 293 cells overexpressing PrPc in the presence of Dox were GRP78, glutathione S-transferase P and alpha-enolase. Besides calcyclin-binding protein, GRP 78 was another calcium binding protein displaying deregulation by overproduction of PrPc. As an endoplasmic reticulum (ER)-resident chaperone, GRP 78 is involved in maintenance of ER homeostasis.39 Interestingly, reduced expression of a large number of genes encoding calcium-binding proteins was detected in brains of sporadic CJD patients.33 As an enzyme involved in protection against oxidative stress, glutathione S-transferase P up-regulation by PrPc implies an increased oxidative stress in PrP+Dox group . Similarly, an increased level of glycolytic enzyme, alpha-enolase, might be seen as a response to the metabolic alterations likely occurring in PrPc-overexpressing cells. A proteomics study of AD brain detected an increased level of alpha-enolase as compared to control brain.40 Two proteins regulated between ctrl-Dox/ PrP+Dox groups belong to proteins related to DNA replication, DNA replication licensing factor MCM3 and glutamate rich WD repeat-containing protein 1. Both proteins were up-regulated by PrP+Dox. Recently, an overexpression of PrPc was shown to induce proliferation and G1/S transition of human gastric cancer cells.41 3. Differentially Expressed Proteins between PrP-Dox/ PrP+Dox Groups. Only two proteins, mitotic checkpoint protein BUB3 and eukaryotic translation initiation factor eIF4H, were detected as significantly regulated between high (PrP-Dox) and moderate level of PrPc expression (PrP+Dox). Mitotic checkpoint protein BUB3 was significantly down-regulated by the high level of PrPc expression. BUB 3 is required for regulation of entry and progression through early stages of mitosis and is essential for prevention of premature sister chromatide separation and missegregation.42 The second protein, eIF4H, was markedly up-regulated by PrP-Dox. The regulation of BUB3 and eIF4H by different levels of PrPc overexpression definitely indicates that PrPc overexpression might be critical for cell cycle regulation and protein synthesis. 4. Differentially Expressed Proteins between Ctrl+Dox/ PrP+Dox Groups. The 2D map comparison between the low level of PrPc expression in the presence of Dox (ctrl+Dox) versus moderate level of PrPc expression in the presence of Dox (PrP+Dox) pointed to regulation of the proteins mainly involved in maintenance of cellular homeostasis. Four proteins exhibited increased expression upon transfection of HEK 293 cells with PRNP gene in the presence of Dox: activator of 90 kDa heat shock protein ATPase homologue 1, ATP synthase subunit alpha (ATPA), elongation factor 1-alpha 1 and Anxa II. Beside NADH dehydrogenase ubiquinone iron-sulfur protein 3, electron transfer flavoprotein subunit beta and ERAB ATPA is the fourth mitochondrial protein shown to be regulated by overexpression of PrPc in this cell culture model. The ATPA is a mitochondrial regulating subunit of the complex V which plays a key role in energy production.43 In AD-affected brain, ATPA exhibited decreased expression44 which may result in disturbance of ATP production and ultimately cell death. Remarkably, the present study revealed ATPA up-regulation by PrP+Dox, as compared to ctrl+Dox, indicating more advantageous energy state of the cells overexpressing PrPc in the presence of Dox as 2692


Ramljak et al. compared to the corresponding control cells. The reason for the observed deviation from the general pattern, suggesting disadvantageous effects upon PrPc overexpression, is not clear. Perhaps, the presence of Dox in the culture medium causes stress which can be better compensated by the cells with moderate level of PrPc expression (PrP+Dox) than by the cells with low PrPc expression level (ctrl+Dox). Annexin II belongs to a family of calcium dependent phospholipid-binding proteins with still undefined cellular function.45 Elongation factor 1-alpha 1 is an evolutionary conserved GTPase that is critically involved in translation fidelity and as a regulator of cytoskeletal rearrangements.46,47 Differential regulation of genes associated with cytoskeletal organization was documented in brains of sporadic CJD.33 The only protein down-regulated by PrP+Dox was NEDD8 conjugating enzyme Ubc 12, a component of NEDD8 pathway known to enhance protein polyubiquitination. In summary, the present examination of the biological processes influenced by PrPc overexpression in HEK 293 cells points to an altered expression of proteins involved in energy and calcium metabolism, maintenance of cellular homeostasis, DNA replication, cytoskeletal organization and cell cycle regulation. Such a variety of biological processes affected by PrPc corroborates its fundamental role in cell physiology. Noteworthy, several proteins found to be regulated by PrPc overexpression in this study were previously described as deregulated in the context of neurodegenerative diseases, but this is the first time that the connection to cellular prion protein was established. The present study revealed a battery of proteins regulated by PrPc-overexpression in HEK 293 cells and as such may serve as a foundation for future studies of the single proteins and their role in exerting damaging effects upon PrPc overexpression. Prnp0/0 Cell Line. An advantageous, neuroprotective effect of PrPc has been reported in wild-type mice as opposed to prion protein deficient mice under various experimental conditions.11–13 Compelling evidence suggests that the introduction of the prion protein gene into PrPc-deficient cells, enduring apoptosis, exerts a beneficial, anti-apoptotic effect.1,48 However, the proteins regulated upon introduction of the prion protein gene into PrPc-deficient environment remain largely unknown. Here, we detected 14 proteins whose expression was modified following transfection of Prnp0/0 cells with PRNP gene. The advantage of this experimental system lies in negative Prnp0/0 background allowing detection of proteins solely influenced by PrPc. Two proteins belonging to the annexin family, Anxa V and Anxa II, were regulated after transfection of PRNP into Prnp0/0 cells. Annexin V, which showed an up-regulation after PrPc expression, is a calcium binding protein that appears to promote survival of developing cortical neurons against peroxide and hypoxia injuries.49 Gene expression profiling in the frontal cortex of sporadic CJD patients revealed an up-regulation of Anxa V.33 In the present study, we showed by Western blot analysis that constitutive overexpression of PrPc in HEK 293 cells leads to an up-regulation of Anxa V as well (Supporting Information Figure 10). The reason that Anxa V regulation was not detected following 2-DE analyses of HEK 293 cells overexpressing PrPc in the Tet-Off system might lie in an insufficient sensitivity of the Coomassie staining method. Surprisingly, although Anxa II showed a prominent up-regulation in PrPcoverexpressing HEK 293 cells, it showed a slight but significant down-regulation after the introduction of PRNP gene into Prnp0/0 cells. Probably, the observed discrepancy in regulation

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Physiological Role of the Cellular Prion Protein (PrPc) c

c

of Anxa II by PrP is caused by different levels of PrP expression and/or presence/absence of endogenous PrPc. Recently, PrPc’s close association with glucose homeostasis was reported.50 Lactate dehydrogenase A, found to be up-regulated following PrPc expression in Prnp0/0 cells, is a glycolytic enzyme playing a significant role in the regulation of the glycolytic pathway. Interestingly, although a marker for cellular damage, the levels of LDH isoenzymes in cerebrospinal fluid of CJD patients exhibited significantly different levels than those seen in other dementias. Hence, LDH isoenzymes were suggested as possible parameters for discrimination between CJD and other dementias.51 In addition to LDH regulation by PrPc in transfected Prnp0/0 cells, HEK 293 cells overexpressing PrPc exhibited an up-regulation of another glycolytic enzyme, alpha enolase (see HEK 293 Cell Line). Remarkably, aldolase C, also implied in glycolytic pathway was recently identified as a novel interaction partner of PrPc19 allowing an assumption that PrPc might have a role in the regulation of glycolytic pathway. Kuwahara and colleagues1 demonstrated for the first time that the introduction of PRNP gene into immortalized hippocampal Prnp0/0 neurons undergoing apoptosis rescues the apoptotic phenotype. Strong evidence indicates that PrPc induces SODC activity, thus, preventing superoxide generation and ultimately apoptosis.48 Our finding that the introduction of PRNP into Prnp0/0 cells markedly stimulates the expression of SODC is in accordance with the above-mentioned reports. Another protein providing protection against apoptotic cell death, Hsp27, was significantly up-regulated upon transfection of Prnp0/0 cells with PRNP gene. This protein has the ability to increase intracellular levels of glutathione and, thus, to reduce the production of reactive oxygen species (ROS).52 In addition, overexpression of Hsp27 prevents apoptosis by directly interacting with cytochrome c.53 An earlier study demonstrated that, under serum deprived conditions, the level of cytochrome c was significantly decreased in mitochondrial fractions of prion protein deficient neuronal cells contributing to apoptotic phenotype. In turn, the reintroduction of PrPc increased cytochrome c level in mitochondrial fractions.54 Taking together the latter findings with our observation that solely an introduction of PrPc into Prnp0/0 cells increases expression of Hsp27, we propose Hsp27 as a possible candidate capable of mitigating apoptosis in PrPc-deficient neuronal cells following PrPc expression. Introduction of PrPc into Prnp0/0 cells led to significant down-regulation of hetereogeneous nuclear ribonucleoproteins (hnRNP) C1/C2. Interestingly, hnRNPA2/B1 was previously described as a PrPc interaction partner.19 hnRNPs are RNA binding proteins contributing to mRNA splicing and its transport from nucleus to cytoplasm.55,56 PrPc octapeptide repeat region displays similarities with the nucleic acid binding and strand-annealing octarepeats of mammalian hnRNP.57 Our finding contributes to accumulating evidence that PrPc may affect nucleic acid metabolism. A key enzyme of the reversible nonoxidative branch of the pentose phosphate pathway, transaldolase, essential for the maintenance of glutathione in the reduced state and thus protection against ROS, was significantly down-regulated following expression of PrPc in Prnp0/0 cells. Reduced levels of transaldolase expression in Jurkat human T cells resulted in increased glucose 6-phosphate dehydrogenase activity and increased glutathione levels leading to inhibition of apoptosis.58 In summary, it is clear that transfection of Prnp0/0 cells with PRNP gene exhibits different proteome changes than the one

c

obtained after PrP -overexpression in HEK 293 cells. The proteins found to be regulated by PrPc expression in Prnp0/0 cells were mainly related to protection against apoptosis. The association of PrPc with the majority of these proteins has not been described so far and might further improve our understanding of the mechanisms supporting the protective role of PrPc.

Conclusions Our study provides additional insight into the physiological function(s) of PrPc via distinct protein profiles caused by the presence/absence and/or different levels of PrPc expression in two cell culture systems. A high proportion of proteins deregulated in PrPc-overexpressing HEK 293 cells was involved in energy metabolism and cellular homeostasis. Hence, the previously reported increased sensitivity of PrPc-overexpressing cells to apoptotic stimuli might be caused by perturbed expression of proteins essential for energy production and maintenance of cellular homeostasis. A particularly significant point of this study is PrPc overexpression-induced regulation of several proteins which play a contributory role to AD and PD pathogenesis. This finding may be helpful in understanding the common molecular mechanisms underlying the pathogenesis of prion diseases and other neurodegenerative disorders. The levels of several other proteins including calcium-related proteins as well as proteins involved in cytoskeleton organization, cell cycle control, protein degradation and nucleic acid metabolism were also altered, confirming a fundamental relevance of PrPc in diverse physiological processes. The introduction of PRNP into Prnp0/0 cells correlated positively to regulation of proteins mainly implied in protection against oxidative stress and apoptosis. Altogether, the presence/ absence and/or the level of PrPc expression seem to be crucial for the fluctuation between PrPc’s pro- and antiapoptotic properties. This study may serve as a foundation for future examinations of the single PrPc-regulated proteins contributing to PrPc’s pro- or antiapoptotic effects.

Acknowledgment. The authors thank Dr. S. Ku¨gler for his valuable advices during cloning experiments, Darinka Petrova for her help regarding densitometric analyses and Joanna Gawinecka for engaging in a workout of additional experiments required for the revised version of the manuscript. The excellent technical support of Christina Wiese, Christa Scholz, Monika Bodemer and Tatjana Pfander is greatly appreciated. This work was in part supported by the Deutsche Forschungsgemeinschaft through DFG Research Center “Molecular Physiology of the Brain”. Supporting Information Available: Figures of Doxinduced post-translational modification of elfin protein, expression of cell type specific markers in Prnp0/0 cell line, and Western blot analysis of Anxa V after constitutive overexpression of PrPc in HEK 293 cells. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kuwahara, C.; Takeuchi, A. M.; Nishimura, T.; Haraguchi, K.; Kubosaki, A.; Matsumoto, Y.; Saeki, K.; Yokoyama, T.; Itohara, S.; Onodera, T. Prions prevent neuronal cell-line death. Nature 1999, 400, 225–226. (2) Bounhar, Y.; Zhang, Y.; Goodyer, C. G.; LeBlanc, A. C. Prion protein protects human neurons against Bax-mediated apoptosis. J. Biol. Chem. 2001, 276, 39145–39149.

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Physiological Role of the Cellular Prion Protein - American Chemical

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