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A ubiquitin variant-based affinity approach selectively identifies substrates of the ubiquitin ligase E6AP in complex with HPV-11 E6 or HPV-16 E6

2020; Elsevier BV; Volume: 295; Issue: 44 Linguagem: Inglês

10.1074/jbc.ra120.015603

1083-351X

Felix A. Ebner, Carolin Sailer, Daniela Eichbichler, Jasmin Jansen, Anna Sladewska-Marquardt, Florian Stengel, Martin Scheffner,

Cancer-related Molecular Pathways

The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP. The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP. Because the coding capacity of viral genomes is rather limited, it is essential for viruses to reprogram the host cell metabolism according to viral need. This type of adaption can be achieved by various means, including modulation of host cell gene expression and exploitation of host cell regulatory proteins. Thus, in-depth analysis of viral regulatory proteins and their effect on host cell metabolism provides an attractive opportunity to obtain insight into mechanisms and processes governing cellular proteostasis. A prominent example is provided by papillomaviruses that contain a genome of ∼8 kb harboring only ∼10 genes (1de Villiers E.M. Fauquet C. Broker T.R. Bernard H.U. Zur Hausen H. Classification of papillomaviruses.Virology. 2004; 324 (15183049): 17-2710.1016/j.virol.2004.03.033Crossref PubMed Scopus (2312) Google Scholar, 2de Villiers E.M. Cross-roads in the classification of papillomaviruses.Virology. 2013; 445 (23683837): 2-1010.1016/j.virol.2013.04.023Crossref PubMed Scopus (336) Google Scholar, 3Doorbar J. Quint W. Banks L. Bravo I.G. Stoler M. Broker T.R. Stanley M.A. The biology and life-cycle of human papillomaviruses.Vaccine. 2012; 30 (23199966): F55-F7010.1016/j.vaccine.2012.06.083Crossref PubMed Scopus (767) Google Scholar, 4Harden M.E. Munger K. Human papillomavirus molecular biology.Mutat. Res. Rev. Mutat. Res. 2017; 772 (28528688): 3-1210.1016/j.mrrev.2016.07.002Crossref PubMed Scopus (76) Google Scholar). Thus, for viral propagation papillomaviruses largely depend on the host cell machinery. Human papillomaviruses (HPVs) have been classified into different genera, with α-papillomaviruses comprising HPV types that infect mucosal and cutaneous epithelia (1de Villiers E.M. Fauquet C. Broker T.R. Bernard H.U. Zur Hausen H. Classification of papillomaviruses.Virology. 2004; 324 (15183049): 17-2710.1016/j.virol.2004.03.033Crossref PubMed Scopus (2312) Google Scholar, 2de Villiers E.M. Cross-roads in the classification of papillomaviruses.Virology. 2013; 445 (23683837): 2-1010.1016/j.virol.2013.04.023Crossref PubMed Scopus (336) Google Scholar, 3Doorbar J. Quint W. Banks L. Bravo I.G. Stoler M. Broker T.R. Stanley M.A. The biology and life-cycle of human papillomaviruses.Vaccine. 2012; 30 (23199966): F55-F7010.1016/j.vaccine.2012.06.083Crossref PubMed Scopus (767) Google Scholar, 4Harden M.E. Munger K. Human papillomavirus molecular biology.Mutat. Res. Rev. Mutat. Res. 2017; 772 (28528688): 3-1210.1016/j.mrrev.2016.07.002Crossref PubMed Scopus (76) Google Scholar). 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This differential association with clinical lesions is also reflected in different biochemical properties of the major HPV oncoproteins, E6 and E7. For example, the high-risk E6 proteins utilize the cellular E3 ubiquitin ligase E6-associated protein (E6AP) to target the tumor suppressor p53 for ubiquitination and degradation (summarized in (7Mantovani F. Banks L. The human papillomavirus E6 protein and its contribution to malignant progression.Oncogene. 2001; 20 (11753670): 7874-788710.1038/sj.onc.1204869Crossref PubMed Google Scholar, 8Scheffner M. Whitaker N.J. Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.Semin. Cancer Biol. 2003; 13 (12507557): 59-6710.1016/S1044-579X(02)00100-1Crossref PubMed Scopus (131) Google Scholar, 9Beaudenon S. Huibregtse J.M. 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Accordingly, a still-increasing number of potential interaction partners of high-risk E6 proteins has been reported, though the physiological relevance of many of these interactions remains unclear, with the exception of p53 and some PDZ domain-containing proteins (for reviews, see (12White E.A. Howley P.M. Proteomic approaches to the study of papillomavirus-host interactions.Virology. 2013; 435 (23217616): 57-6910.1016/j.virol.2012.09.046Crossref PubMed Scopus (53) Google Scholar, 13Wallace N.A. Galloway D.A. Novel functions of the human papillomavirus E6 oncoproteins.Annu. Rev. Virol. 2015; 2 (26958922): 403-42310.1146/annurev-virology-100114-055021Crossref PubMed Scopus (36) Google Scholar)). Furthermore, most of the proteins reported to bind to high-risk E6 proteins do not appear to interact with low-risk E6s (12White E.A. Howley P.M. Proteomic approaches to the study of papillomavirus-host interactions.Virology. 2013; 435 (23217616): 57-6910.1016/j.virol.2012.09.046Crossref PubMed Scopus (53) Google Scholar, 13Wallace N.A. Galloway D.A. Novel functions of the human papillomavirus E6 oncoproteins.Annu. Rev. Virol. 2015; 2 (26958922): 403-42310.1146/annurev-virology-100114-055021Crossref PubMed Scopus (36) Google Scholar). A remarkable exception is E6AP. E6AP is encoded by the UBE3A gene (14Sutcliffe J.S. Jiang Y.H. Galijaard R.J. Matsuura T. Fang P. Kubota T. Christian S.L. Bressler J. Cattanach B. Ledbetter D.H. Beaudet A.L. The E6-AP ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region.Genome Res. 1997; 7 (9110176): 368-37710.1101/gr.7.4.368Crossref PubMed Scopus (80) Google Scholar, 15Yamamoto Y. Huibregtse J.M. Howley P.M. 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Increased gene dosage of Ube3a results in autism traits and decreased glutamate synaptic transmission in mice.Sci. Transl. Med. 2011; 3: 103-19710.1126/scitranslmed.3002627Crossref Scopus (176) Google Scholar). Redirecting of E6AP by high-risk E6 proteins to substrates that in the absence of E6 are not targeted by E6AP, such as p53, is assumed to contribute to HPV-induced cervical carcinogenesis (8Scheffner M. Whitaker N.J. Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.Semin. Cancer Biol. 2003; 13 (12507557): 59-6710.1016/S1044-579X(02)00100-1Crossref PubMed Scopus (131) Google Scholar, 9Beaudenon S. Huibregtse J.M. HPV E6, E6AP and cervical cancer.BMC Biochem. 2008; 9 (19007434): S410.1186/1471-2091-9-S1-S4Crossref PubMed Scopus (98) Google Scholar). Indeed, the presence of E6AP is essential for HPV-induced cervical carcinogenesis in a transgenic mouse model (23Shai A. Pitot H.C. Lambert P.F. 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Association of E6AP (UBE3A) with human papillomavirus type 11 E6 protein.Virology. 2007; 358 (17023019): 303-31010.1016/j.virol.2006.08.038Crossref PubMed Scopus (56) Google Scholar, 11Kuballa P. Matentzoglu K. Scheffner M. The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins.J. Biol. Chem. 2007; 282 (17085449): 65-7110.1074/jbc.M605117200Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), it is tempting to speculate that E6AP is a critical interaction partner of low-risk and high-risk E6 proteins and is utilized by these to target an overlapping spectrum of host proteins for ubiquitination. Yet, in particular, the potential targets of the low-risk E6-E6AP ubiquitin ligase complex remain largely elusive (13Wallace N.A. Galloway D.A. Novel functions of the human papillomavirus E6 oncoproteins.Annu. Rev. Virol. 2015; 2 (26958922): 403-42310.1146/annurev-virology-100114-055021Crossref PubMed Scopus (36) Google Scholar). In principle, there are at least two main possibilities to identify substrate proteins of ubiquitin ligases at the proteome level (summarized in (25Iconomou M. Saunders D.N. Systematic approaches to identify E3 ligase substrates.Biochem. J. 2016; 473 (27834739): 4083-410110.1042/BCJ20160719Crossref PubMed Scopus (81) Google Scholar)). Substrates can be identified by manipulating the expression levels of the respective ubiquitin ligase or of a subunit of a ubiquitin ligase complex, in this case E6 proteins, in cells (e.g. (26Burande C.F. Heuzé M.L. Lamsoul I. Monsarrat B. Uttenweiler-Joseph S. Lutz P.G. A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation.Mol. Cell. 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Although subsequent affinity-based purification procedures allow the identification of proteins whose ubiquitination status is affected by the presence or absence of the E6 proteins, it remains unclear whether the proteins identified represent direct substrates of the E6 proteins or whether their ubiquitination status is indirectly affected. Alternatively, potential substrate proteins can be identified in vitro (25Iconomou M. Saunders D.N. Systematic approaches to identify E3 ligase substrates.Biochem. J. 2016; 473 (27834739): 4083-410110.1042/BCJ20160719Crossref PubMed Scopus (81) Google Scholar), for instance by using whole-cell extracts. The advantage of a whole-cell extract system is that the ubiquitin machinery can be manipulated such that ubiquitination mainly depends on the activity of the ubiquitin ligase of interest, thereby minimizing the possibility that the ubiquitination status of a protein is indirectly affected. In any case, results obtained in any system have to be validated in vitro using recombinant proteins and in cell culture experiments. To identify potential substrate proteins of both low-risk and high-risk E6 proteins, we established an affinity-based enrichment approach using whole-cell extracts and a dedicated biotin-tagged ubiquitin variant that in the absence of the E6 proteins is only poorly used by E6AP for ubiquitination. Enriched proteins were subsequently identified by high-resolution MS (LC–MS/MS) and label-free quantification. We further validated E6-mediated ubiquitination/degradation of some of the proteins identified both in vitro by using recombinant proteins and within cells by transient transfection experiments, demonstrating the potential of this approach. To enable purification and identification of potential substrates of the E6 proteins with high specificity (for a scheme of the general workflow, see Fig. 1A), we envisioned that a ubiquitin-based affinity approach should fulfill the following criteria. First, ubiquitin needs to be equipped with an affinity tag that does not interfere with E6-E6AP–mediated ubiquitination and allows enrichment of ubiquitinated proteins under harsh conditions to minimize the possibility of (co)purifying nonubiquitinated proteins. Second, E6AP was reported to catalyze mainly the formation of Lys-48–linked ubiquitin chains (poly-ubiquitination) (29Wang M. Pickart C.M. Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis.EMBO J. 2005; 24 (16341092): 4324-433310.1038/sj.emboj.7600895Crossref PubMed Scopus (100) Google Scholar, 30Kim H.C. Huibregtse J.M. Polyubiquitination by HECT E3s and the determinants of chain type specificity.Mol. Cell Biol. 2009; 29 (19364824): 3307-331810.1128/MCB.00240-09Crossref PubMed Scopus (173) Google Scholar). Furthermore, poly-ubiquitination itself can render the identification of the respective protein by LC–MS/MS difficult, because the high number of ubiquitin-derived peptides poses a challenge to detect less-prominent peptides. Thus, a ubiquitin variant that is not or only poorly used by E6AP for poly-ubiquitination should prove helpful in circumventing this drawback. Third, the approach should depend on the presence of E6 proteins to increase the likelihood that the proteins identified are indeed substrates of the E6-E6AP complex rather than E6AP alone. A possibility to achieve this is the use of ubiquitin variants that are efficiently used by the E6-E6AP complex but not by E6AP alone, such as defined hydrophobic patch mutants (31Mortensen F. Schneider D. Barbic T. Sladewska-Marquardt A. Kühnle S. Marx A. Scheffner M. Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26216987): 9872-987710.1073/pnas.1505923112Crossref PubMed Scopus (39) Google Scholar). To address the first criterion, we adapted a published procedure that results in site-specific modification of ubiquitin at Lys-6, presumably because of the catalytic microenvironment of the ε-amino group of K6 (32Macdonald J.M. Haas A.L. London R.E. Novel mechanism of surface catalysis of protein adduct formation. NMR studies of the acetylation of ubiquitin.J. Biol. Chem. 2000; 275 (10906321): 31908-3191310.1074/jbc.M000684200Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 33Shang F. Deng G. Liu Q. Guo W. Haas A.L. Crosas B. Finley D. Taylor A. Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation.J. Biol. Chem. 2005; 280 (15790562): 20365-2037410.1074/jbc.M414356200Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To biotinylate ubiquitin, we employed sulfo-N-hydroxysuccinimide long-chain biotin (Fig. S1B), and MS analysis of the resulting product revealed that ubiquitin was indeed primarily biotinylated at Lys-6 (Fig. S1, C and D). Because E6AP mainly catalyzes the formation of Lys-48–linked ubiquitin chains (29Wang M. Pickart C.M. Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis.EMBO J. 2005; 24 (16341092): 4324-433310.1038/sj.emboj.7600895Crossref PubMed Scopus (100) Google Scholar, 30Kim H.C. Huibregtse J.M. Polyubiquitination by HECT E3s and the determinants of chain type specificity.Mol. Cell Biol. 2009; 29 (19364824): 3307-331810.1128/MCB.00240-09Crossref PubMed Scopus (173) Google Scholar), we expected that biotinylation of ubiquitin at Lys-6 should not, or only moderately, interfere with E6AP activity. To determine whether this is the case, we performed E6AP auto-ubiquitination assays (34Nuber U. Schwarz S.E. Scheffner M. The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate.Eur. J. Biochem. 1998; 254 (9688277): 643-64910.1046/j.1432-1327.1998.2540643.xCrossref PubMed Scopus (98) Google Scholar) and ubiquitination assays with whole-cell extracts derived from HEK293T cells (35DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.Mol. Cell Biol. 1987; 7 (3031469): 379-38710.1128/mcb.7.1.379Crossref PubMed Scopus (892) Google Scholar) that express high levels of WT p53. Indeed, E6AP was efficiently auto-ubiquitinated in the presence of biotinylated ubiquitin (Fig. S2A). Similarly, addition of HPV-16 E6 and E6AP to HEK293T cell extracts resulted in quantitative poly-ubiquitination of p53 under the conditions used. The ubiquitinated forms of p53 were enriched by incubation with streptavidin beads and subjected to Western blotting analysis using a p53-specific antibody (Fig. S2B). However, LC–MS/MS analysis of the eluates did not result in the identification of a significant number of p53-derived peptides (not shown). To address the second criterion, we resorted to a ubiquitin variant in which lysine residues 48 and 63 are replaced by arginine (Ub-K48/63R), because this variant should only be poorly used by E6AP for poly-ubiquitination. Surprisingly, it turned out that Ub-K48/63R is not only poorly used by E6AP for poly-ubiquitination (Fig. 1B) but for ubiquitination in general (Fig. S3, A–C). Moreover, Ub-K48/63R is efficiently used by the E6-E6AP complex (Fig. 1D and Fig. S3). In a simplified view, E6AP-mediated ubiquitination is a two-step process. In the first step, E6AP forms a thioester complex with ubiquitin, and in the second step, E6AP catalyzes the covalent attachment of ubiquitin to substrate proteins by isopeptide bond formation. Thioester complex formation assays with WT ubiquitin and with Ub-K48/63R clearly showed that E6AP can readily form thioester complexes with either of these and that the efficiency is not affected by E6 (Fig. S3D). This strongly indicates that the E6 proteins rescue the inability of E6AP to transfer Ub-K48/63R to substrate proteins, which is reminiscent of the results we previously obtained with hydrophobic patch mutants of ubiquitin (31Mortensen F. Schneider D. Barbic T. Sladewska-Marquardt A. Kühnle S. Marx A. Scheffner M. Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (26216987): 9872-987710.1073/pnas.1505923112Crossref PubMed Scopus (39) Google Scholar). Because E6 also does not affect the lysine residues of ubiquitin used by E6AP for poly-ubiquitination (Fig. 1C), Ub-K48/63R fulfills not only the second but also the third criterion. Thus, Lys-6–biotinylated Ub-K48/63R is ideally suited to identify proteins that are targeted by the E6-E6AP complex, but not by E6AP alone. To identify substrate proteins of HPV-16 E6 (high risk) and HPV-11 E6 (low risk), we employed whole-cell extracts derived from HaCaT cells (36Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J. Cell Biol. 1988; 106 (2450098): 761-77110.1083/jcb.106.3.761Crossref PubMed Scopus (3334) Google Scholar). HaCaT cells are spontaneously immortalized keratinocytes, and because keratinocytes constitute the host cells of HPVs, they represent an appropriate means to obtain first insights into the substrate pattern of HPV E6 proteins. HaCaT cell extracts were incubated in the presence of recombinant ubiquitin-like modifier–activating enzyme 1 (UBA1), ubiquitin-conjugating enzyme UbcH7, biotinylated Ub-K48/63R, and E6AP in the absence and presence of GST fusion proteins of HPV-16 E6 or HPV-11 E6. Ubiquitinated proteins were enriched by affinity chromatography using streptavidin beads and upon elution separated by SDS-PAGE (Fig. 2A). After cutting the gel in slices and in-gel tryptic digestion, the samples were analyzed by LC–MS/MS followed by label-free quantification. In total, 1509 proteins were identified in at least two of three biological replicates (HPV-16 E6, HPV-11 E6, and control reaction in the absence of any E6), and the corresponding intensities are shown as a heat map in Fig. 2B. Of these, 199 and 179 proteins were significantly enriched by (false discovery rate (FDR) = 0.001, s0 = 2, n = 3, two-sample t test) and, thus, identified as potential substrate proteins of HPV-16 E6 and HPV-11 E6, respectively, with 109 of these shared by both E6 proteins (Fig. 3C and Supporting data 1; a gene ontology annotation analysis is shown in Fig. S4). The corresponding volcano plots are shown in Fig. 3, A and B.Figure 3Analysis of potential substrate proteins of HPV-16 E6 and HPV-11 E6. A and B, volcano plots of proteins that were significantly enriched in the presence of the E6 proteins and thus represent potential substrate proteins of HPV-16 E6 and HPV-11 E6, respectively. Significant enrichment was determined relative to proteins identified in the reactions in the absence of E6 proteins (Supporting data 1). Plotted is the log2 fold change versus the negative logarithm of the p-values. Black dots indicate significant enrichment (FDR = 0.001, S0 = 2, n = 3, two-sample t test). Blue dots/protein names indicate significant enrichment of previously reported substrate proteins or interaction partners of the E6 proteins or of E6AP (i.e. RAD23A). Red dots/protein names indicate proteins that were not reported previously to represent substrates of the E6 proteins and were selected for validation. C, heat map of proteins significantly enriched for both HPV-16 and HPV-11 E6 and HPV-16 E6 or HPV-11 E6.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Closer inspection of the results obtained by LC–MS/MS showed that for 67 (HPV-16 E6) and 44 (HPV-11 E6) of the enriched proteins at least one peptide harboring a diGly motif was identified (Supporting data 1), strongly indicating that the lysine residue within the respective peptide was ubiquitinated (37Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21 (12872131): 921-92610.1038/nbt849Crossref PubMed Scopus (1249) Google Scholar). Furthermore, streptavidin beads were stringently washed upon incubation with the ubiquitination reaction mixtures under conditions (6 m urea and 6 m guanidinium chloride) that in general result in protein denaturation but do not interfere with the biotin-streptavidin interaction (38González M. Argaraña C.E. Fidelio G.D. Extremely high thermal stability of streptavidin and avidin upon biotin binding.Biomol. Eng. 1999; 16 (10796986): 67-7210.1016/S1050-3862(99)00041-8Crossref PubMed Scopus (133) Google Scholar). Taken together, our data strongly indicate that the vast majority of the enriched proteins identified were indeed ubiquitinated. 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