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J. Biol. Chem., Vol. 279, Issue 24, 25729-25744, June 11, 2004
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From the Department of Biochemistry and Microbiology, Center for Molecular Biology and Gene Therapy, Loma Linda University School of Medicine, Loma Linda, California 92354
Received for publication, February 3, 2004 , and in revised form, March 4, 2004.
| ABSTRACT |
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16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway. | INTRODUCTION |
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In previous work, our laboratory found that E6 could protect cells from TNF (37) and that it does so by binding to the C-terminal end of TNF R1, thus blocking transmission of the apoptotic signal from TNF R1 through the TNF R1-associated death domain (TRADD) (23). These observations led us to ask whether E6 might also modulate apoptotic signaling triggered by related mechanisms, such as the Fas pathway. Apoptosis triggered through the Fas pathway begins with engagement of Fas, either by Fas ligand or with appropriate anti-Fas antibodies, allowing the formation of a death-inducing signaling complex (DISC). Fas first recruits Fas-associated death domain (FADD) to bind to its own death domain (DD) by way of the DD present on FADD. FADD contains both a DD and a death effector domain (DED) and, therefore, functions as an adaptor molecule by also binding to the DED of pro-caspase 8 via a DED/DED interaction. Aggregation of the pro-caspase 8 molecules leads to their mutual activation and the consequent engagement of the caspase cascade, resulting in apoptosis (see Refs. 38-45 for reviews). FADD may be required for the function of additional signaling pathways as well (46), including the mediation of TRAIL-induced apoptosis (47-50) and the induction of caspase-independent, necrotic cell death (51).
In this study, we found that HPV 16 E6 protects cells from Fas-triggered apoptosis and that it does so by binding to FADD and accelerating its degradation. Because FADD is known or speculated to participate in a number of apoptotic pathways, these observations suggest that E6 may function to protect expressing cells from apoptosis triggered by a variety of ligands and may therefore play an important role in helping cells infected with HPV 16 avoid elimination by host defense mechanisms and thus contribute to the survival and propagation of the virus.
| EXPERIMENTAL PROCEDURES |
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Cell CultureU2OS cells, derived from a human osteosarcoma, were obtained from the ATCC (Manassas, VA). They were cultured in Mc-Coy's 5A medium (Invitrogen) supplemented to contain 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml), and streptomycin (100 µg/ml) (Sigma).
PlasmidsThe pHA-E6 S and pHA-E6 AS plasmids have been described previously (23), and respectively contain either the sense or the antisense versions of epitope-tagged E6 (HA-E6) under the control of the CMV promoter.
The pTet-Off plasmid coding for tetracycline activator, pTRE-luc, coding for luciferase under the control of the tetracycline activator, pTK-Hyg, coding for hygromycin resistance, as well as the cloning plasmid pTRE2, were obtained from and described in the Tet-Off kit from Clontech. pTRE-HA-E6 was obtained by cloning the HindIII blunt end-BamHI fragment from pHA-E6 S into the EcoRI blunt end-BamHI sites of the pTRE2 vector.
pcDNA3-FADD was kindly provided by Dr. Carl Ware (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and served as the basis for all our additional FADD-expressing constructs. The pM-FADD and pVP-FADD plasmids needed to perform the mammalian two-hybrid analysis (Clontech) were obtained by PCR amplification of FADD using primers 5'-GACCCGTTCCTGGTGCTG-3' (forward) and 5'-ATGCCTGTGGTCCACCAGC-3' (reverse). This PCR product was then cloned into pCR-Blunt II-Topo using the Zero Blunt Topo PCR cloning kit (Invitrogen), and the EcoRI-EcoRI fragment subcloned into the pM DNA-BD and pVP AD EcoRI sites. Positive and negative control plasmids for this system were either provided in the kit (Clontech) or described earlier (23). pFLAG FADD was obtained by cloning the FADD HindIII-XbaI fragment from pTopoFADD into the pFLAG-myc-CMV-22 vector (Sigma).
To express FADD and its variants in both the Escherichia coli system and in the in vitro transcription/translation system (T7 reticulocyte) (Promega), the EcoRI-blunt end-XhoI fragment from pM-FADD, coding for FADD, was cloned in-frame with the His6 epitope of pTriEx-4 (Novagen) by using the SmaI-XhoI sites of its multiple cloning site to produce the plasmid pHis-FADD. A similar approach was used to produce the two deletion fragments. To produce deletion D1 (lacking amino acids 23-62), the SacI-SacI fragment was removed from the FADD coding sequence. To produce deletion D2 (lacking amino acids 1-79), the SalI-blunt end-XhoI fragment from pM-FADD was cloned into the SmaI-XhoI sites of pTriEx-4. The point mutations were created in a D1 background using the QuickChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The sequences of the primers used for mutagenesis are presented in Table I.
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pHA-E6large produces a transcript (
480 bp) that cannot be spliced to yield the short processed message, and thus codes only for the large, full-length version of HA-E6. The construct was produced using pHA-E6 S as a template and then mutagenizing the donor splice site from AGGT to AGAT (52) by using the QuickChangeTM mutagenesis kit (Stratagene). In contrast, pHA-E6small produces only the short transcript and thus codes only for the short, half-length version of HA-E6. It was produced by isolating RNA from the stable cell line U2OSE612 (23) and then performing RT-PCR by using 5'-ATGATATCCTATCCATACGATGTT-3' and 5'-TGGGTTTCTCTACGTGTTCTTGAT-3', which correspond to the beginning and end of both the short and long versions of E6. The PCR product of 0.2 kb was then cloned into pCR2.1 (Invitrogen), and its EcoRI-EcoRI fragment was then subcloned into the EcoRI site of the pE-CMV-1 vector (23).
TransfectionsTransfections were carried out using FuGENE 6 (Roche Applied Science), as directed by the manufacturer. For transient transfections, cells were analyzed 40-48 h post-transfection. For stable transfections, clones were passaged into selection medium containing G418 (500 µg/ml) or hygromycin (100 µg/ml). Following 3 weeks of selection, individual clones were grown and analyzed for protein expression by immunoblotting and/or RT-PCR.
To normalize the transfection efficiency for the FADD degradation experiment, pCMV-SEAP was co-transfected with pcDNA3-FADD. The media were changed 48 h post-transfection. After an additional 3 h of incubation, during which SEAP was secreted into this fresh media, 20 µl of the conditioned media was analyzed for SEAP activity by using the Great EscAPe chemiluminescence detection kit (Clontech) according to the manufacturer's protocol. A normalized amount of lysate, corresponding to 13 relative light units as measured on a ML3000 microtiter plate luminometer (Dynatech Laboratories), was then loaded per lane for separation by SDS-PAGE.
ImmunoblottingCells (106) were lysed in 100 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 10 min on ice. One tablet of protease inhibitor mixture (Roche Applied Science) per 10 ml of buffer was added just prior to use. The protein concentration in cleared lysates was measured using the Bio-Rad Protein Assay (Bio-Rad). Lysates (40 µg total protein/lane) were then subjected to 12% SDS-PAGE and transferred to Immobilon P membranes (Millipore Corp.). After treating membranes with 5% milk in TBST (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), rat anti-HA peroxidase-conjugated antibodies (Roche Applied Science), anti-FADD antibodies (Santa Cruz Biotechnology), anti-GST monoclonal antibodies (Santa Cruz Biotechnology), or anti-
-actin monoclonal antibodies (Sigma) were applied at a 1:1000 dilution (for the anti-
-actin antibodies, a 1:5000 dilution was used) and allowed to incubate for 2 h, with rocking, at room temperature. After washing with TBST, peroxidase-coupled secondary antibodies were added for the anti-FADD, anti-GST, or anti-
-actin detections. In the case of anti-FADD, anti-rabbit antibodies (Sigma) were used, whereas in the case of anti-GST and anti-
-actin, anti-mouse antibodies (Pierce) were applied (1:2000 or 1:5000 dilution) and allowed to incubate with rocking at room temperature for 30 min. Membranes were then washed again with TBST. Detection of the protein was performed by using the chemiluminescent SuperSignal West Femto or Pico Maximum Sensitivity substrate (Pierce).
ImmunoprecipitationCells (2-5 x 106) were lysed in 500 µl of the immunoblotting lysis buffer, and cleared lysates were incubated with 2 µg of monoclonal anti-HA antibody (Roche Applied Science) at 4 °C for 1 h with rotation. Protein A-agarose slurry (50 µl) (Santa Cruz Biotechnology) was added to each lysate, and lysates were incubated for 3 h at 4 °C. The protein A slurry was then washed three times with lysis buffer, followed by one wash with high salt buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 1 µM dithiothreitol) and one wash with the same buffer lacking NaCl. The precipitates were fractionated by 12% SDS-PAGE, and immunoblotting was performed as described above.
Treatment of Cells with Anti-Fas, Mitomycin C, MNNG, Ceramide, MG-132, and CycloheximideTo measure cell survival following anti-Fas treatment, cells were seeded into 96-well plates (1 x 104 cells/well) and allowed to adhere overnight. Anti-Fas (at the concentrations noted in text and figures) was then added in the presence of cycloheximide (5 µg/ml), and the cells were incubated for 16 h prior to measuring the number of viable cells by the MTT assay (described below).
To determine the ability of cells to accumulate increased levels of p53 following DNA damage, cells were treated with mitomycin C and assayed for the resulting p53 levels. Cells were seeded into 24-well plates (1 x 105 cells/well) and allowed to adhere for
24 h, treated with mitomycin C for 16 h, harvested, and the lysates analyzed for p53 by ELISA (described below).
To determine cell survival following MNNG or ceramide treatment, cells were seeded into 96-well plates (2 x 104 cell/well, 50 µl total volume) and allowed to adhere overnight. For the ceramide experiments, the medium was then removed and replaced with medium supplemented to contain 2% serum. Ceramide was then added to the indicated final concentration, and the cells were incubated for 24 h prior to measurement of the number of viable cells by the MTT assay. For the MNNG experiments, MNNG was added to a final concentration of 25 µg/ml for 4 h prior to harvest and MTT assay.
To examine the amount of FADD in cells expressing E6, U2OSE6tet24 cells were seeded into 6-well plates (2 x 105 cells/well) in the presence of the indicated concentrations of doxycycline. Following an overnight incubation, the cells were transfected with pcDNA3-FADD, and 40 h post-transfection were treated with MG-132 (50 µM) or cycloheximide (5 µg/ml) for the indicated time. Cells were harvested, and the lysates were analyzed by immunoblot using
-FADD antibodies.
Cell Viability AssayFollowing treatment of cells as described above, the incubation medium was removed and exchanged with 80 µl of fresh medium. Twenty microliters of MTT was then added (5 mg/ml stock), and cells were incubated at 37 °C for 3 h. The medium was removed, and 150 µl of Me2SO was added and allowed to incubate for 10 min. The solution was mixed by pipetting, and the absorbance of each well was measured at 490 nm.
Development and Use of the Tet-off SystemU2OS cells capable of expressing variable amounts of HA-E6, regulated by the dose of drug present in the media, were created using the Tet-Off system (Clontech) following the manufacturer's protocol with some modifications. Cultures of these cells were grown in the indicated concentrations of doxycycline for 2 days prior to use, and the indicated concentrations of doxycycline were maintained in the media throughout each experiment.
p53 ELISACells were assayed for p53 by ELISA essentially as described previously (23, 53). The monoclonal antibody pAB122 (hybridoma obtained from ATCC; antibodies were purified from the culture medium using protein A-Sepharose) was used as the primary or capture antibody, and biotinylated anti-p53 (Roche Applied Science) was used as the detection antibody, and glutathione S-transferase-p53 (Santa Cruz Biotechnology) was used as a standard.
Reverse Transcriptase-PCRThe reverse transcriptase-PCR was used to analyze E6 transcripts in cell lines stably transfected with E6 AS, E6 S, E6large, and E6small. Three and a half micrograms of total RNA was isolated from each cell line using the TRIzol reagent (Invitrogen) and used as a template. cDNA was synthesized by using SuperScriptTM II reverse transcriptase (Invitrogen) and an oligo(dT) primer (Amersham Biosciences). The primers 5'-ATGATATCCTATCCATACGATGTT-3' and 5'-TGGGTTTCTCTACGTGTTCTTGAT-3' were then used to amplify the PCR products from the cDNA, using one-twentieth of the total cDNA reaction mixture. To control for possible contamination by genomic DNA, parallel reactions were run using 0.175 µg of total RNA in the absence of the reverse transcriptase enzyme. Reaction mixtures were separated on a 4.5% NuSieve GTG-agarose gel (FMC BioProducts).
Caspase 3 and 8 AssaysCells were plated onto 100-mm plates at a density of 2-5 x 106 cells/plate and incubated overnight. Anti-Fas (50 ng/ml) was added, along with cycloheximide (5 µg/ml). Cells were harvested by scraping with a rubber policeman at designated intervals and were lysed in 160 µl of Caspase 3 Lysis Buffer (Sigma). Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad).
Caspase 3 activity was measured using the Caspase 3 Colorimetric Assay kit (Sigma) as directed by the manufacturer. Cell lysates (25 µl) were incubated with substrate (Ac-DEVD-pNA) in the presence or absence of the caspase 3 inhibitor Ac-DEVD-CHO. Absorbance at 405 nm was determined
3 h following initiation of the reaction. The activity in wells treated with inhibitor was subtracted from the activity in untreated wells, and the activity was normalized to the amount of protein present in each sample. Caspase 3 activity in treated samples was expressed as a percentage of caspase 3 activity in the untreated parental cells. Three plates of treated and untreated cells were measured for each time point.
Caspase 8 activity was measured using the Caspase 8 Assay kit (Calbiochem) according to the manufacturer's instructions, using 50 µl of cell lysate per well and using IETD-pNA as the colorimetric substrate, and in the presence or absence of the caspase 8 inhibitor Ac-IETD-CHO. Absorbance at 405 nm was determined
6 h following initiation of the reaction, and calculations were performed as described for the caspase 3 assay.
Mammalian Two-hybrid AssayThe mammalian two-hybrid binding assay was performed according to the manufacturer's instructions (Clontech). The indicated combinations of vectors were transfected into U2OS cells (5 x 105/well, 6-well plates) along with the chloramphenicol acetyltransferase (CAT)-expressing reporter plasmid using FuGENE 6 (Roche Applied Science) as directed by the manufacturer. Forty eight h following transfection, CAT activity was measured colorimetrically using a commercially available CAT-ELISA kit (Roche Applied Science) as directed by the manufacturer.
Expression and Purification of Recombinant ProteinsTo purify the GST, GST-E6, and GST-E6small proteins, cleared lysates of transformed isopropyl-1-thio-
-D-galactopyranoside-induced E. coli (BL21 (DE3) pLysS, Novagen) cultures (from 100 ml of LB broth) were incubated with 1 ml of glutathione beads (Sigma) pre-equilibrated in lysis buffer (50 mM Tris-HCl, 0.5 M NaCl, 10 mM EDTA, 10 mM EGTA, 10% glycerol, 1% Triton X-100, 20 µg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride, 1 tablet of protease inhibitor (Roche Applied Science), pH 8.0). After incubation for 1 h on a rotator at 4 °C, the beads were washed with lysis buffer, followed by three washes with 10 ml of wash buffer (25 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 5% glycerol, pH 7.5). Following the last wash, beads with their bound GST and GST-E6 proteins were resuspended in PBS supplemented with 10% glycerol and 2 mM DTT.
The His-FADD, His-D1, His-D2, His-M3, and His-QQQ proteins, as well as those resulting from the other point mutations, were purified from E. coli lysates (from a 500-ml culture) using the B-PER His6 Fusion Protein Purification kit (Pierce) according to the manufacturer's protocol. All proteins were soluble in the original elution buffer (which contains 250 mM imidazole). Following dialysis against a buffer of 20 mM HEPES, pH 7.4, 137 mM NaCl, 2 mM KCl, 5% glycerol, and 1 mMDTT, the His-D1, His-D2, His-M3, and other point mutant proteins remained soluble, although the full-length His-FADD protein precipitated. The expression of full-length His-FADD was achieved using the T7 TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's protocol. All proteins were separated by SDS-PAGE and stained with Coomassie Blue. The protein concentration for each preparation was estimated by using the Bio-Rad Protein Assay.
In Vitro Pull-down AssaysAn in vitro pull-down approach was used to measure the ability of bead-bound GST or GST-E6 proteins (glutathione beads obtained from Sigma) to bind to either FADD or FLAG-FADD in mammalian cell lysates or to the transcription/translation product of full-length His-FADD. Cells (5 x 106) transfected 48 h previously with the desired expression plasmid were lysed in 500 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 µM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor tablet/10 ml buffer (Roche Applied Science)), and the cleared cell lysate (or, in the case of the transcription and translation product, 10 µl of reticulocyte lysate) was incubated with 30 µl of either GST or GST-E6 beads (in PBS plus 2 mM DTT) on a rotator for 2 h at 4 °C. Three washes were then done with lysis buffer, followed by one wash in high salt buffer and a final wash in buffer lacking NaCl as described under "Immunoprecipitation." Bound proteins were eluted in 30 µl of 2.5x SDS loading buffer and fractionated on 12% PAGE. Following transfer to polyvinylidene fluoride membranes (Millipore), FADD was detected using anti-FADD polyclonal antibodies (Santa Cruz Biotechnology).
To analyze the ability of bead-bound GST, GST-E6, and GST-E6small to bind to purified His-FADD D1, His-FADD D2, His-FADD M3, His-FADD QQQ, and the other point mutants, the slurry (40 µl) was incubated with 6 µg of the indicated version of FADD in 500 µl of 20 mM HEPES, pH 7.4, 137 mM NaCl, 2 mM KCl, 2 mM DTT, and 5% glycerol for 2 h at 4 °C with rotation. Beads were then washed three times with the above buffer with the addition of 0.1% Nonidet P-40 (20 min, 4 °C, rotation). Bound proteins were eluted in 30 µl of 2.5x SDS loading buffer and fractionated on 12% PAGE. Following transfer to polyvinylidene fluoride membranes (Millipore), FADD was detected using anti-FADD polyclonal antibodies (Santa Cruz Biotechnology). The same membrane was then stripped and reblotted with anti-GST monoclonal antibodies to demonstrate the presence of the GST and GST-E6 proteins. In addition, 0.6 µg of the FADD D1 and D2 proteins was separated by SDS-PAGE and stained by Coomassie Blue to show the loaded protein.
| RESULTS |
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-actin, using amounts of lysate corresponding to the input of the top two panels in Fig. 2A, showed no change. To verify that decreases in HA-E6 were continuing to occur, even at the higher levels of doxycycline, RT-PCR was used to detect the E6 transcript in these cells under these conditions. The results are shown in Fig. 2A, 3rd panel, and demonstrate that decreases in the amount of message continue to occur at the higher levels of doxycycline. They also demonstrate that even at a relatively high level of doxycycline (200 ng/ml), some HA-E6 message is still being made. An RT-PCR analysis of the actin message, used for normalization, is shown in Fig. 2A, bottom panel.
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The next step was to test the sensitivity of the U2OSE6tet24 and U2OSE6tet26 cells to anti-Fas when grown in the presence of different concentrations of doxycycline. As shown in Fig. 2C, as the level of dox decreased from 100 to 0.4 ng/ml and the level of HA-E6 rose, the viability of cells exposed to anti-Fas (50 ng/ml) increased from about 30 (U2OSE6tet24) or 43% (U2OSE6tet26) to
60%. There was no significant difference between the response of the two clones. These data provide strong evidence that not only does E6 expression protect cells from Fas-mediated apoptosis but that this protection is dose-dependent and rises with increasing amounts of E6.
HPV 16 E6 Inhibits Activation of Caspase 3 and Caspase 8 If HPV 16 E6 does protect cells by inhibiting one or more of the upstream steps in the Fas pathway, it should interfere with the activation of caspases 3 and 8. To test this prediction, U2OS cells transfected with either the sense (U2OSE612) or the antisense (U2OSE6AS) version of HPV 16 E6 were treated with
-Fas and then analyzed for the activation of the two caspases using colorimetric assays that respond to cleavage of the appropriate substrate. Fig. 3A shows that whereas cells expressing the antisense version of E6 respond to anti-Fas treatment with a robust increase in caspase 3 activation within 4.5 h, cells transfected with the sense version of E6 (U2OSE612) do not. The results from the caspase 8 experiment (Fig. 3B) were similar with respect to the trend but to a lesser degree. These results indicate that E6 acts on the Fas-mediated pathway at or prior to activation of these two caspases and suggest that it may function by interacting with the DISC.
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The E6 Gene Produces Three Messages and Two ProteinsThe E6 gene produces at least three messages because of the differential splicing of mRNA (52, 54) (Fig. 6A). The large transcript (
0.48 kb) codes for the full-length protein of
16 kDa, whereas the two smaller transcripts (of 0.3 and 0.2 kb) code for very similar proteins of approximately half the length of the longer E6. To determine whether the regions of E6 retained in the small version were sufficient for binding to FADD, constructs that coded exclusively for either the short or the long version were created and used to analyze the FADD binding and protective ability of the two E6 proteins. To determine which transcripts were actually generated in expressing cells, the various versions of E6 were stably transfected into cells and the transcripts examined using RT-PCR (Fig. 6B). As anticipated, cells transfected with the intact gene express both the large and small transcripts, whereas cells given only the large or small gene express only the expected transcript. To determine whether the protein associated with the two shorter transcripts of 0.3 and 0.2 kb (approximately a 7-8-kDa protein) was actually made, the vector expressing the HA-tagged version of the small protein was transiently transfected into U2OS cells, and the lysates were analyzed by immunoblot (Fig. 6C). In addition to the expected band at
7 kDa, some faster migrating species were also observed and may represent degradation products.
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Together, these data demonstrate that E6 binds to FADD without the requirement of additional cellular factors, that amino acids 23-62 and all those from 79 to 208 are dispensable for this binding, and that a major contribution to this binding is made by serines 10, 14, 16, and 18 and/or glutamic acid 19. These results therefore provide a molecular explanation for the protection from Fas-mediated apoptosis provided to cells by E6.
HPV 16 E6 Mediates the Rapid Degradation of FADDHPV 16 E6 mediates the rapid degradation of some, but not all, of the proteins to which it binds, leading to a decrease in the steady-state level of those proteins. In previous work, our laboratory found that whereas HPV 16 E6 bound to TNF R1, it did not affect its level or accelerate its degradation (23). To determine whether E6 affected the steady-state level of FADD, U2OS and U2OSE6tet24 cells were transiently transfected with pcDNA-FADD, coding for FADD, and the U2OSE6tet24 cells were incubated in the presence of increasing amounts of doxycycline, thus experiencing the expression of decreasing amounts of HA-E6. Normalized amounts of lysates were then analyzed by Western blot using antibodies directed against FADD. As shown in Fig. 9A, when little or no E6 was expressed (lanes 2 and 7), FADD was readily detected. However, as the level of E6 was increased by lowering the amount of doxycycline present in the media (Fig. 9A, lanes 6, 5, 4, and 3), the level of FADD decreased. In principle, these results could be due either to an E6-mediated increase in FADD degradation or to a decrease in FADD transcription. If an increase in proteosome-mediated degradation were the mechanism, one prediction is that the inclusion of a protease inhibitor, such as MG 132, would attenuate this loss. Performing this experiment demonstrated that when MG 132 was added to U2OSE6tet24 cells in the absence of dox (hence expressing high levels of E6), the amount of cellular FADD did indeed increase (Fig. 9B). A second prediction is that if protein synthesis were blocked with cycloheximide, the levels of FADD would decrease more rapidly in the presence of E6 than in its absence. The results shown in Fig. 9C demonstrate that this is, in fact, the case. In the presence of 500 ng/ml dox (and absence of E6), very little FADD is lost within a 90-min incubation following cycloheximide addition, whereas in the presence of 0.4 ng/ml dox (and hence the presence of E6), the level of FADD decreases by
50%. Together, these results provide strong evidence that HPV 16 E6 can indeed lower cellular levels of FADD and that it does so by accelerating the degradation of FADD.
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| DISCUSSION |
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The ability of human papillomaviruses to modulate host apoptotic responses has recently been gaining recognition (reviewed in Ref. 66). For example, some studies have found that cervical carcinoma cells positive for HPV 16 and HPV 16-immortalized nonmalignant human keratinocytes are relatively resistant to apoptosis induced by Fas L (67, 68), although these studies did not identify the particular viral genes responsible. Other studies have focused on the roles specific genes may play in immune evasion. For example, the E5 protein can reduce the amount of the CD95 receptor at the cell surface (69), and both the E6 and E7 genes have been reported to modulate the cellular response to various apoptotic signals, although the reported effects differ, with some groups reporting sensitization (70-73) and others reporting protection (23, 37, 74). It may well be that the effect of E6 and E7 on a given cell will depend on the cellular environment as well as on the nature of the apoptotic signal. Our previous finding that HPV 16 E6 could protect cells from TNF-mediated apoptosis (23) led to the speculation that it could also block apoptotic signaling through the closely related Fas pathway. Treatment of E6-expressing stable transfectants with anti-Fas led to the conclusion that E6 could in fact protect cells from Fas-mediated apoptosis (Fig. 1). The mechanism for this protection was then examined and led to the finding that HPV 16 E6 binds to FADD, accelerates its degradation, and thus makes it unavailable for the transduction of apoptotic signals.
The development of an inducible system for E6 expression, in which the amount of E6 could be regulated by varying the amount of doxycycline present in the media, allowed confirmation of this protective effect of E6 and the determination that it was dose-dependent, with higher levels of E6 providing greater levels of protection, at least up to some point (Fig. 2). This is consistent with a model in which HPV 16 E6 binds to FADD and mediates its degradation (Fig. 9); as the level of E6 increases, correspondingly increasing numbers of FADD molecules can be targeted for degradation. The data from the stable clones (Fig. 1B) suggest that at very high levels of E6, likely higher than those achieved with the tet-regulated system, the protective effect of E6 may be lost, perhaps because these higher amounts of E6 participate in additional interactions that cancel out its protection. Another possibility, of course, is that these clones with very high levels of E6 may not be entirely representative, due to additional and uncharacterized events. Most interesting, even at high (200 µg/ml) levels of doxycycline, some HA-E6 message was being produced, presumably leading to protein production, although the level is too low to allow detection by immunoblot (Fig. 2A). This may be due to the regulatory environment of the genomic sites into which the constructs integrated and may explain why, even at high levels of doxycycline, the U2OSE6tet24 and U2OSE6tet26 cells experience some protection from anti-Fas as compared with the U2OS cells (Fig. 2C, far left data point).
Both genotoxic damage (initiated by MNNG) and ceramide lead to apoptosis through the mitochondrial pathway, which joins the receptor-mediated pathways at the level of caspase 3 activation. Each of these agents was able to efficiently initiate cell death in U2OSE6tet24 cells, regardless of the concentration of doxycycline in which they were cultured (Fig. 4). This meant that E6 targets the apoptosis signal at or upstream of caspase 3 activation in the Fas pathway. This is consistent with the findings that both caspase 3 and caspase 8 activities were diminished in cells expressing E6 (Fig. 3) and supports a model in which E6 interacts with the DISC. It is unclear why greater increases of caspase 3 than of caspase 8 were observed following treatment with anti-Fas, as both caspases are expected to function in the death pathway. One factor may be that in our hands the caspase 3 assay appears to be more sensitive than the caspase 8 assay. Alternatively or in addition, these findings suggest the possibility that players in this pathway may have connections and interactions that are not accounted for in a simple, unidirectional model (75, 76).
A molecular explanation for this decreased apoptotic signal was provided by the finding that E6 binds to FADD. This binding was demonstrated by the mammalian two-hybrid system and three types of in vitro pull-down assays (Figs. 5 and 8). Using purified proteins (GST-E6 and His-tagged versions of FADD) (Fig. 8) allowed the conclusion that the binding of E6 to FADD was direct and did not require additional factors. In addition, the use of the different variants of FADD (D1, D2, and M3) allowed us to localize the region(s) of FADD required for binding to E6 to the N terminus of FADD and implicated amino acids 10, 14, 16, 18, and/or 19. It has been reported previously that both Fas- and TNF-mediated apoptosis use the same binding surface of FADD to trigger signal transduction and that binding surface includes helices
2 and
3 of the FADD death domain (77). However, the implicated stretch required for binding to E6 is N-terminal to these two helices, suggesting that the E6-binding site is distinct from the sites required for interactions with their apoptotic signaling partners. Although E6 binds to both TNF R1 and to FADD, it does not indiscriminately interact with all apoptotic signaling molecules. For example, it binds only weakly to TRADD (Fig. 5A and data not shown), demonstrating specificity for its binding partners.
Including FADD, the number of identified E6-binding cellular proteins has now reached nearly two dozen, and the molecular basis for these protein-protein interactions is beginning to be understood. Two E6-binding motifs have thus far been identified. One occurs in the PDZ domain of proteins such as hDlg, hScrib, MAGI-1, and MUPP1 and binds to the ETZV sequence at the far C terminus of E6 (reviewed in Ref. 12). The presence of this PDZ ligand domain is likely important in the targeting of these proteins for degradation by E6 (78). The second motif is known as the L2G box and was initially identified by Elston and co-workers (79), who used a yeast two-hybrid system to screen a 16-mer peptide library for peptides that specifically interact with HPV 16 E6. From this work, they identified an E6-binding motif of (E/D)L(L/V)G, which was also found in the known E6-binding proteins E6-AP and E6-BP. Further work (80) has established that the actual consensus sequence is LhX
Lsh (where h is an amino acid residue capable of accepting hydrogen bonds (Asp, Glu, Gln, or Asn), X refers to any amino acid,
is hydrophobic, and s denotes a small amino acid residue such as G or A) and that this region must be helical to bind to E6. It is found in a number of cellular targets for E6, including E6AP, hMcm7, E6BP, paxillin, and TNF R1 (16, 23, 79, 81). However, it is worth noting that the classical L2G box motif is not present in the death effector domain of FADD, which does bind to E6, and mutagenesis of the three copies of an abbreviated version ((E/D)LL) did not diminish the binding activity. Our identification of the four serines and/or one glutamic acid as important for binding affinity (Figs. 7 and 8) suggests the presence of a novel E6-binding motif present in FADD, possibly interacting with a different region or face of the E6 protein. Further work will be required to further define and establish a consensus for this new motif.
The fact that more than one transcript is produced from the E6 genes of oncogenic strains of HPV has been known for some time (52, 54, 82, 83). Initially, it was thought that the alternatively spliced versions of the E6 message might function to enhance the translation efficiency of E7 (52), but more recent work suggests that the shorter E6 protein is actually produced and may function by binding to and inhibiting the biological activities of the full-length protein. For example, the truncated version of HPV 18 E6 can bind to the full-length versions of HPV 18 E6, HPV 16 E6, and E6AP, inhibits (full-length) E6-mediated degradation of p53, and increases E6-mediated transcription and growth arrest in E6-expressing cells (82). It has also been shown that the ratio of the full-length and small versions of E6 may vary in cytologically abnormal cells (84), and this observation may be linked to the ability of the short E6 to interact with proteins such as E6 and E6-AP (66). We have been able to demonstrate production in cells of both the message and protein for the small E6 protein, and we have shown that the large and small versions differ in function, in that only the large version is capable of binding to FADD and protecting cells from Fas (Fig. 6).
It is intriguing that E6 accelerates the degradation of FADD (Fig. 9) although not of TNF R1, reflecting the variation seen in the fate of many of the proteins to which E6 binds. In this respect, it may be relevant to note that whereas E6 binds to the DD of TNF R1, it binds to the DED of FADD even though FADD contains both a DD and a DED. Whereas these two regions do share some homology, they differ sufficiently to be considered distinct motifs. Understanding the molecular basis for this difference in the fate of bound proteins will be an important area for future investigation.
This degradation of FADD is likely to have biological consequences. It has been reported, for example, that loss of FADD expression is correlated with the development of tumoral status in thyroid follicular cells (85) and that inhibition of FADD expression using antisense technology reduced keratinocyte apoptosis triggered by UV irradiation (86) as well as a failure of carboplatin-induced cell death in human tongue carcinoma cells (87). In our system, of course, the E6-mediated degradation of FADD may also contribute to the resistance of cells to TNF, as FADD participates in both pathways.
Papillomaviruses are persistent viruses that remain in their hosts for long periods. The papillomavirus-specific immune response is either weak or undetectable, and little or no inflammatory response is elicited by papillomavirus infection. The capacity of E6 to protect cells from Fas and from TNF may be an important factor in this lack of a host inflammatory response and thus contribute both to the persistence of this virus and to its oncogenic potential. Moreover, the findings by our laboratory and others that many important signaling and regulatory proteins in cells, such as FADD, can bind to E6 suggest additional approaches for the development of therapeutic agents for cervical cancer. For example, it has been reported recently that when RNAi was applied to HeLa cells, which express HPV 18 E6 and E7 genes, the cells underwent senescence (88) and that apoptosis could be induced in HPV-positive cells by peptide aptamers targeting E6 (89). Other groups (90) have focused downstream of E6