Comparative Proteomic Analysis Implicates COMMD Proteins as

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Comparative Proteomic Analysis Implicates COMMD Proteins as EpsteinBarr Virus Targets in the BL41 Burkitt’s Lymphoma Cell Line Zacharati Gkiafi and George Panayotou* Institute of Molecular Oncology, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece

bS Supporting Information ABSTRACT: EpsteinBarr virus (EBV) infection is a major health problem associated with a variety of diseases, including Burkitt’s lymphoma. EBV promotes its effects through the activation of multiple signaling pathways, with NF-kB mediated transcription being a major target. We have undertaken a comparative proteomic approach using 2D-electrophoresis and mass spectrometry to identify EBV-regulated proteins in the BL41 Burkitt’s lymphoma cell line. Many of the proteins differentially regulated were previously known mediators of EBV action. Among the novel proteins identified, three members of the conserved COMMD family were found to be down-regulated. Further analysis of this family at the mRNA level, using reversetranscriptase or real-time PCR, showed that 7 out of 10 COMMD members were affected in EBV-transformed BL41 cells. Since COMMD family proteins have been implicated as negative regulators of the NF-kB transcription factor, our data are consistent with a hypothesis that EBV down-regulates COMMD proteins in order to enhance NF-kB mediated transcriptional events and B-lymphocyte transformation. KEYWORDS: EpsteinBarr virus, B-lymphocytes, Burkitt’s lymphoma, proteomics, COMMD

’ INTRODUCTION EpsteinBarr virus (EBV) is a human herpes virus that mainly infects B cells and is the causative agent of infectious mononucleosis, while also contributing to the development of malignant tumors, such as Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and others.1 EBV was first identified in Burkitt’s lymphoma, but its precise role in the development of this disease remains unclear after more than 50 years of research. EBV infection is characterized by different latency types, which are dependent on the expression of distinct sets of EBV genes.2,3 LMP1, an integral membrane protein, is the major oncogene of EBV and can transform resting B-lymphocytes into lymphoblastoid cell lines (LCLs). LMP1 resembles a constitutively active CD40 receptor and transmits sustained proliferation and survival signals in B cells. LMP1 signals via the NF-kB transcription factor through two distinct pathways, the canonical and the noncanonical, but can also trigger the activation of other signaling pathways, such as p38, c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K/Akt).48 The delineation of the molecular events that follow EBV infection and their role in cell immortalization and cancer formation is of great importance in understanding EBV-related diseases and in the development of novel therapeutic strategies. In order to investigate the molecular mechanisms that regulate B cell transformation by EBV latency type III infection, we employed a proteomic approach on two well characterized cell lines: BL41, a non-infected Burkitt’s lymphoma cell line, and BL41/B95.8, a BL41 derivative infected with the B95.8 strain of EBV.912 The EBVpositive cell line expresses the full array of EBV latent genes (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP1, LMP2A r 2011 American Chemical Society

and LMP2B), indicative of latency type III.13,14 This cell system has previously been used to analyze differential gene expression with cDNA microarrays.11,13 Other proteomic1517 or microarray1821 approaches have focused on different cell systems, primarily lymphoblastoid cell lines, while the role of EBV in other malignancies, such as nasopharyngeal or gastric carcinoma, has also been investigated with similar tools, including a phosphoproteome analysis.22 Using two-dimensional electrophoresis, we identified many differentially expressed proteins, which were classified into distinct categories according to their molecular function. A substantial proportion of these have previously been shown to be EBV-regulated proteins. Interestingly, among the novel proteins identified, three members of the COMMD family were shown to be down-regulated in the EBV-infected cells, a finding confirmed also at the mRNA level and extended to an additional four members. The involvement of this protein family in the regulation of NF-kB mediated transcription may provide interesting new insights in the molecular mechanisms underlying the transformation of B-lymphocytes by EBV.

’ MATERIALS AND METHODS Cell Lines

BL41, BL41/B95.8, and IB4 cells were provided by Dr. G. Mosialos, and BL31/B95.8 cells by Dr. Rowe Martin. Healthy B-lymphocytes were a gift of Dr. K. Stamatopoulos. Lysates from purified B cells and EBV-infected LCLs (35 days post infection) Received: July 30, 2010 Published: May 26, 2011 2959

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Journal of Proteome Research from the same donor were obtained from Prof. Micah Luftig, Duke University Medical Center. Cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin (Gibco), and 1% GlutaMax (Gibco), in an atmosphere containing 5% CO2 at 37 °C. Sample Preparation for Proteomic Analysis

Cell suspensions were centrifuged at 1200 rpm for 5 min. The pellet was washed twice with cold PBS and lysed in 50 mM Tris pH 7.4, 2% CHAPS, 15 mg/mL DTT, protease inhibitor cocktail (1:40) (Roche), at 4 °C for 10 min. The lysate was centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant was acetoneprecipitated for 16 h at 20 °C. Treatment with benzonase (Merck) was used to remove nucleic acids from cell extracts and proteins were again acetone-precipitated and stored at 80 °C until use. Nuclear extracts from B cells were prepared as described previously with modifications.23 Cells were washed with PBS and pelleted at 1000 rpm at 4 °C for 5 min. The pellet was resuspended in 4 volumes of hypotonic lysis buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 5 mM DTT, 0.5 mM PMSF, 0.5% Nonidet P-40 and protease inhibitor cocktail (Roche)), for 1 h at 4 °C. Cells were then homogenized by 2030 strokes in a prechilled glass Dounce homogenizer (B-type pestle) and then centrifuged for 5 min at 2000 rpm at 4 °C to separate nuclei from the cytoplasmic fraction. Collected nuclei were washed with 1 mL of buffer A, centrifuged for 5 min at 2000 rpm at 4 °C, resuspended in 3 volumes of high-salt buffer B (20 mM HEPES pH 7.9, 20% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF, 5 mM DTT and protease inhibitor cocktail) and incubated on ice for 30 min to extract proteins from nuclei. The homogenate was then centrifuged for 20 min at 13,000 rpm at 4 °C. The supernatant was collected, acetone-precipitated for 16 h at 20 °C and stored at 80 °C until use. Protein concentrations were determined by Bradford assay. Two-Dimensional Electrophoresis (2DE)

Protein pellets were resuspended in 340 μL of rehydration buffer (7 M urea, 2 M Thiourea, 2% CHAPS, 10 mg/mL DTT, 2% IPG buffer and traces of bromophenol blue), and the sample was loaded on 18 cm immobilized pH gradient strips of wide (310 NL) and narrow range (35.6 NL, 47, 4.55.4, 58, 5.36.5, 6.27.5, 711 NL) (GE Healthcare and Bio-Rad). Sample uptake was achieved after 1416 h at 20 °C without applying any current, and isoelectric focusing was carried out on a MultiPhor II apparatus (Pharmacia Biotech) or Protean IEF Cell (Bio-Rad). For SDS-PAGE, the IPG strips were incubated in equilibration buffer containing 50 mM Tris-HCl, pH 6.8, 6 M urea, 1% SDS, 30% glycerol, traces of bromophenol blue and 7.5 mg/mL DTT for 15 min and then incubated for 15 min in equilibration buffer supplemented with 45 mg/mL iodoacetamide. The equilibrated IPG strips were placed on top of a 10% or 12% SDS-PAGE (20 cm 20 cm) and sealed with 0.5% molten agarose. Electrophoresis was carried out using Protean II xi 2-D system (Bio-Rad) in a tris-glycine SDS buffer, at 30 mA per gel. Silver Staining and Spot Quantitation

Silver staining was performed according to published protocols24 with minor modifications. Briefly, the gels were fixed in 50% methanol/5% acetic acid for 20 min, washed for 10 min with 50% methanol, and washed in water for a minimum of 2 h. The gels were subsequently sensitized with a 1 min incubation in 0.02% sodium thiosulfate and rinsed with distilled water twice for 1 min each. After incubation with 5 μg/mL DTT for 30 min, a chilled 0.1% cold silver nitrate solution was added for 20 min.

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The gels were then rinsed twice with water for 1 min and developed with a 0.04% formalin solution in 2% sodium carbonate with shaking. Color development was terminated by discarding the reagent and adding 5% acetic acid. Silver-stained gels were stored in a solution of 1% acetic acid at 4 °C until analyzed. In total, 62 pairwise comparisons between the two lines were performed. Specifically, 24 pairwise comparisons were performed on 310 NL IPG strips and 17 pairwise comparisons were performed on 47 NL IPG strips. For 11 of the above comparisons separate nuclear/cytoplasmic fractions were used, while for the rest total lysates were applied. Total lysates were also used for 5 pairwise comparisons on 3.05.6 NL IPG strips, 2 pairwise comparisons on 4.55.4 IPG strips, 1 on 58 IPG strips, 3 on 5.36.5 IPG strips, 5 on 6.27.5 IPG strips, and 5 on 7.011.0 NL IPG strips. Out of the total 62, 42 gels were biological replicates and 20 were technical replicates. Gels were manually inspected and scanned on a GS-800 densitometer (Bio-Rad), and digital images were analyzed with the image analysis software PDQuest (version 8.0; Bio-Rad). After spot detection and background subtraction, quantitative determination of matched spot volumes was performed. Normalization of each individual protein spot quantity was made according to the total quantity of the valid spots on the gel. In-Gel Protein Digestion

In-gel digestion was performed as described previously.25 Protein spots were excised from 2DE gels and destained with 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. They were then rinsed with water, cut into small pieces, dehydrated with acetonitrile and incubated in 50 mM ammonium bicarbonate/50% (v/v) acetonitrile. The gel pieces were dried under vacuum and reswelled with a 12 ng/μL solution of Gold Trypsin (mass spectromentry grade, Promega) in 50 mM ammonium bicarbonate at 4 °C for 30 min. Excess trypsin was removed, and the gel pieces were incubated in 50 mM ammonium bicarbonate at 37 °C overnight. Following enzymatic digestion, peptides were extracted from the gel in sequential washes of 50 mM ammonium bicarbonate, 100% acetonitrile, 5% formic acid and 100% acetonitrile. The pooled peptide extracts were dried under vacuum and finally resuspended in 10 μL of 2% acetonitrile/0.1% formic acid). Peptide Separation and MS/MS Analysis

Nanohigh performance liquid chromatography (nano-HPLC) was used for separation of tryptic peptides (Ultimate, LC Packings) with a PepMap reversed phase C18 column (75 μm 15 cm, Dionex). Peptides were eluted at a flow-rate of 200 nL/min with a 280% (v/v) acetonitrile/water gradient containing 0.1% formic acid, over 35 min. Eluting peptides were introduced online into an LCQ Deca ion-trap mass spectrometer equipped with a nanoelectrospray source (ThermoFinnigan). For the MS/ MS analysis of peptides, a top-three method was employed, using the software Xcalibur for data collection. During the first scanning event, spectra representing mass to charge ratios (m/z) between 500 and 2000 amu were collected. Next, the three most abundant ions from the first scanning event were subjected to MS/MS analysis. Dynamic exclusion for 0.5 min was enabled after three MS/MS scans of the same precursor ion. The MS raw file was searched using the SEQUEST algorithm against a merged and indexed IPI human (3.55) and EBV1/EBV2 protein database. The search parameters were fully trypsin specificity, two missed cleavage sites, peptide tolerance of 2.0 amu and fragment ion tolerance of 1.0 amu. Possible modifications due to 2960

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Journal of Proteome Research

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methionine oxidation and cysteine carbamidomethylation were also included in the search. The criteria for positive identification of peptides were Xcorr >1.5 for singly charged ions, Xcorr >2.0 for doubly charged ions, and Xcorr >2.5 for triply charged ions with Delta Correlation Score (DelCn) of 0.1 or higher. Finally, for each identified protein the predicted or published molecular weight was checked against the one observed on the gels. Western Blot Analysis

Samples containing the same total amount of protein were separated on SDS-PAGE gels and transferred to nitrocellulose membrane (Whatman). Membranes were blocked with 1%BSA in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20, for 1 h at room temperature. After blocking, membranes were incubated with antibodies for 16 h at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies (antimouse, Southern Biotech; antigoat, Santa Cruz; antirabbit, Millipore) for 1 h at room temperature. The following antibodies were used: GAPDH (6C5, Santa Cruz), p100/p52 (C-5, Santa Cruz), CD40 (C-20, Santa Cruz), COMMD9 (E-14, Santa Cruz), LMP1 (S12, a gift from Dr. G. Mosialos), COMMD1 (Abnova), vimentin (Sigma), c-Jun (Cell Signaling), tubulin (Sigma), TFIIB (C-18, Santa Cruz), EBNA-2 (PE2, Abcam), and 14-3-3ε (T-16, Santa Cruz). Detection was carried out using ECL-Plus reagents (Amersham). Visualization was carried out using either the chemiluminescence detection mode of a Storm phosphoimager (Molecular Dynamics) or Fuji X-ray film. Digital images were analyzed for changes in signal intensity with the image analysis software ImageQuant (version 5.0) or PDQuest. DNA Extraction, RNA Isolation, and RT-PCR Analysis

DNA was extracted from 11.5 107 cells according to Laird et al.26 after proteinase K digestion. The clonal relationship between EBV positive and negative cell lines was tested as described18 with the amplification of the third framework region of the immunoglobulin heavy chain gene, with the only modification that PCR products were analyzed on agarose gels. Total RNA was extracted from cells using TRI Reagent (Applied Biosystems) according to the manufacturer’s instructions. Yield and purity were determined by measuring A260/280 of RNA diluted in RNA storage solution buffer (Applied Biosystems, Ambion). RT-PCR was performed using GoTaq polymerase (Promega) and PCR products were analyzed on agarose gels. Loading control PCR reactions were carried out using primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). COMMD levels were normalized versus GAPDH and the ImageQuant 5.0 software was used to quantitate scanned images. The ratio between the levels in BL41/B95.8 and BL41 cells was calculated and statistical significance was evaluated with a onesample t test versus the hypothetical value of 1 (no change). All primers used in PCR analysis are presented as Supporting Information in Table S3. Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) was performed on diluted cDNA (3 μL, 1:20 dilution) using Sybr Green (Invitrogen) in a Chomo4 real-time PCR detector (Bio-Rad). PCR cycling included an initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s, annealing temperature according to each gene primer set for 10s and amplification at 72 °C, with a plate read at the end of each cycle. The amplification time depended on the sequence length and was calculated according to the DNA polymerase amplification rate (1 min/kb). The reactions were performed in duplicates or in triplicates, and three independent

Figure 1. Characterization of the cell lines used for proteomic comparisons. Western blotting analysis was performed using specific antisera against LMP1 (A), EBNA2 (C), CD40 (D), and p52 (E). GAPDH and TFIIB were used as loading controls. RT-PCR for LMP1 mRNA is shown in panel B. PCR analysis of the FR3-JH region to prove the same clonal origin of BL41 and BL41/95.8 cells is shown in panel F.

experiments were performed. All primers used are presented as Supporting Information in Table S3. Relative accumulation of the COMMD genes normalized against GAPDH was determined from the Ct values as described.27 PCR efficiency of each reaction was calculated according to Ramakers et al.28

’ RESULTS In order to investigate proteome changes induced by EBV transformation, we employed the well-characterized BL41 cell line, originating from an EBV-negative Burkitt’s lymphoma, and compared it to BL41/B95.8, derived from BL41 by in vitro infection with EBV. BL41/B95.8 is characterized as Latency III and expresses the full array of EBV latent genes (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP1, LMP2A and LMP2B).2,9,10,12,13 The same system has previously been used to analyze differences in gene expression using cDNA microarrays,11,13 while in a recent proteomic study, the BL41 line has been compared to IARC-171, an EBV-infected lymphoblastoid line isolated from the same patient as BL41.16 Verification of the maintenance of the EBV viral genome was done by monitoring the levels of the EBV proteins, LMP1 and EBNA2. Cell extracts were prepared from both cell lines and analyzed by Western blotting with specific anti-LMP1 and anti-EBNA2 antisera, confirming its presence only in the EBV transformed cells (Figure 1A and C). Analysis at the mRNA level, also confirmed the exclusive expression of LMP1 in the EBV(þ) cells (Figure 1B). Two further markers of functional EBV transformation were evaluated: Increased levels of CD40 have previously been reported in Burkitt’s lymphoma cell lines 2961



Figure 2. Representative examples of 2D gel features showing differences between the two cell lines. Arrows point to the identified proteins.

and EBV associated tumors, together with other B-cell activation markers, such as CD23 and CD39, as well as adhesion molecules (ICAM1, LFA1 and LFA3).2,7,11 EBNA3B has also been shown to induce the expression of CD40.2,29 Moreover, enhanced production of the NF-kB subunit p52, via increased processing of p100, and concomitant translocation to the nucleus together with RelB are also known effects of EBV infection.5 Figure 1D demonstrates a robust increase of CD40 protein, while Figure 1E shows increased levels of the p52 subunit in the BL41/B95.8 line, compared to BL41. Finally, the same clonal origin of the EBVpositive and negative BL41 cell lines was confirmed by PCR genomic amplification of the FR3-JH region of the immunoglobulin heavy chain gene. Figure 1F shows the same products for the two lines, which are clearly different from those of IB4 (an LCL) and BL31/B95.8 cells (EBV-positive BL line). Having confirmed the correct characteristics of the two cell lines, we proceeded with the comparison of expressed proteins using two-dimensional electrophoresis. In order to reduce sample complexity and increase the number of proteins separated and identified, we employed both subcellular fractionation of the cells in order to enrich cytoplasmic and nuclear fractions, as well as narrow-range isoelectric focusing (IEF) gels, in addition to whole cell lysates and broad range IEF. In total, 62 pairwise comparisons between the two lines were performed. Representative examples of differences observed in 2DE gels are shown in Figure 2. Differentially stained proteins, evaluated by the PDQuest software, were then excised, digested with trypsin, and identified by mass spectrometry. Table 1 summarizes the results, with 19 proteins found to be up-regulated and 19 down-regulated in response to EBV infection (further details for each identification available as Supporting Information, Tables S1 and S2). A fold change is also given for each protein identified. Almost half of the protein spots showed a qualitative difference, i.e., were detected only in one of the two cell lines, so a fold change could not be estimated.

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Confirming the validity of our experimental approach, several proteins previously known to be regulated by EBV were found in our study. For example, vimentin is a 54-kDa cytoskeletal protein that shows a significant increase in expression upon latent infection by EBV. Verification of this result by Western blotting is shown in Figure 3A. The upregulation of vimentin has also been shown in the proteomic study of Brennan et al.16 Additional known proteins include annexin VI, a Ca2þ binding protein;32,33 LDH-A, which is one of the two subunits that constitutes the LDH enzyme, converting pyruvate to lactate, and is elevated in Burkitt’s lymphoma;34 Interferon-induced GTP-binding protein Mx1, which is stimulated by a variety of viral infections35 and was also identified in cDNA microarray studies,11,18,20 as was phosphoglycerate kinase 1 (PGK1);13 PI-9 or serpin B9, which is highly expressed in EBV-transformed lymphoblastoid cell lines and inactivates granzyme B, a proteolytic enzyme that induces apoptosis;36 UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1), which maintains a free ubiquitin pool by processing pro-Ub and rescuing Ub from lysosomal degradation and is detected at higher levels in Burkitt’s lymphoma compared to LCLs;37 CDK4, the core protein controlling the G1/S checkpoint, previously shown to be regulated by LMP1 and EBNA2;38 the La antigen (Lupus La protein), a nuclear phosphoprotein that plays a role in the correct folding and maturation of RNA polymerase III transcripts and has been shown to be present in the cytoplasm of EBV-infected lymphoblastoid cell lines at a higher level than in EBV-negative cell lines;39 actin, also identified in other proteomic studies;16 stathmin-1, an important regulator of cell cycle progression and proliferation, which is phosphorylated in response to EBV and is also regulated by LMP1.16,19,40 Downregulation of stathmin may indeed reveal a change in the stability of microtubules during and after immortalization, as suggested by Toda and Sugimoto41 and confirmed by other studies.16 Although generally there was concordance between our results and those of previous studies, some exceptions were seen, such as endoplasmin, which was found downregulated in our study but up-regulated in Brennan et al.,16 and D3-phosphoglycerate dehydrogenase, for which the opposite was true. Several of the differentially expressed genes identified previously in microarray studies11,13,18,20 have also been confirmed by our proteomic analysis, including vimentin, actin, PGK1, 14-3-3ε, PAF acetylhydrolase subunit γ, Mx1, ezrin, RANBP1 and EEF1R, although two showed the opposite effect (fructose biphosphate aldolase A and ANP32A). Using a specific antibody, we have further validated the difference in expression of 14-3-3ε, an isoform of the phospho-serine binding 14-3-3 protein family (Figure 3A). The rest of the proteins reported in Table 1 have not previously been implicated in lymphocyte transformation or EBV infection. Among these proteins, some are commonly found in many comparative proteomic studies30,31 and therefore their significance is questionable, e.g., fructose biphosphate aldolase A, peroxiredoxin-6, R-enolase, and γ-enolase. However, some of the established targets of EBV infection mentioned above also belong to this list of generally detected proteins, e.g., vimentin, annexin VI, ezrin, and stathmin-1. We therefore cannot exclude a role for a few additional such proteins with functions that may implicate them indirectly in EBV transformation. Examples include HSPA8 (Hsc70) and HSP27, which can interact with EBNA5;42 S100A9, a calcium-binding protein, which may be involved in cell cycle progression;43 cofilin, an actin binding protein, which is regulated by viruses;44 and proteasome subunit 2962



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Table 1. Proteins Up- or Down-Regulated in Response to EBV Infection up-regulated by EBV protein

accession number (IPI)

no. of unique peptides

coverage (%)

fold change

Acidic leucine-rich nuclear phosphoprotein 32 family member A Actin, cytoplasmic 1a,b

IPI00025849.1

7

27.3

c

IPI00021439.1

4

26.1

5.7

R-Enolasea,b

IPI00465248.5

9

29.9

2.4

Annexin VI isoform 2a,b Cell division protein kinase 4a D-3-Phosphoglycerate dehydrogenase Elongation factor 1-R-2 Elongation factor 1-Rb γ-Enolaseb Glucose-6-phosphate isomerasea Interferon-induced GTP-binding protein Mx1a Isoform 1 of heat shock cognate 71 kDa proteina,b Lupus La proteina Phosphoglycerate kinasea,b Ran/TC4 Ribose-5-phosphate isomerase Septin-11 Serpin B9a Vimentina,b

IPI00002459.4 IPI00007811.1 IPI00011200.5

21 1 8

37.8 4.0 23.3

c c 4.4

IPI00014424.1 IPI00025447.8 IPI00216171.3 IPI00027497.5 IPI00167949.6

6 5 3 14 19

16.2 15.2 12.7 29.4 41.1

c c 2.8 6.0 c

IPI00003865.1

16

42.1

c

IPI00009032.1 IPI00169383.3 IPI00643041.3 IPI00026513.6 IPI00019376.6 IPI00032139.1 IPI00418471.6

1 5 9 9 6 4 15

3.4 9.3 44.9 38.3 22.1 21.5 40.6

1.7 9.5 c c c 7.4 c

GO analysis (molecular function) protein binding

ATP binding, nitric-oxide synthase binding, structural constituent of cytoskeleton magnesium ion binding, phosphopyruvate hydratase activity, serine-type endopeptidase activity, transcription corepressor activity, transcription factor activity calcium ion binding, calcium-dependent phospholipid binding ATP binding, cyclin-dependent protein kinase activity, protein binding NAD or NADH binding, electron carrier activity, phosphoglycerate dehydrogenase activity GTP binding, GTPase activity, protein binding, translation elongation factor activity GTP binding, GTPase activity, translation elongation factor activity magnesium ion binding, phosphopyruvate hydratase activity cytokine activity, glucose-6-phosphate isomerase activity, growth factor activity GTP binding, GTPase activity, protein binding ATP binding, ATPase activity, protein binding mRNA binding, nucleotide binding, protein binding, tRNA binding ATP binding, phosphoglycerate kinase activity GTP binding, GTPase activity, protein binding ribose-5-phosphate isomerase activity GTP binding, protein binding protein binding, serine-type endopeptidase inhibitor activity protein binding, structural constituent of cytoskeleton

down-regulated by EBV accession number (IPI)

no. of unique peptides

coverage (%)

fold change

14-3-3 protein εa,b Cofilin-1b COMM domain-containing protein 2 COMM domain-containing protein 3 COMM domain-containing protein 9 Deoxycytidine kinasea

IPI00000816.1 IPI00012011.6 IPI00456048.4

2 7 3

7.0 34.9 30.2

9.1 d 1.2

enzyme binding actin binding protein binding

IPI00015773.3

3

21.5

4.0

protein binding

IPI00305212.5

1

10.6

d

protein binding

IPI00020454.1

5

21.5

3.5

Endoplasmin

IPI00027230.3

22

30.1

5.0

Ezrina,b Fructose-bisphosphate aldolase Ab Heat shock protein beta-1a,b Isoform 1 of L-lactate dehydrogenase A chaina Isoform 1 of protein SET

IPI00843975.1 IPI00465439.5

11 2

23.1 6.3

d d

IPI00025512.2 IPI00217966.7

10 7

68.3 27.4

d d

IPI00072377.1

6

28.3

d

Peroxiredoxin-6b Platelet-activating factor acetylhydrolase IB subunit γa Proteasome subunit R type-5b Protein S100-A9b Similar to Ran-BP1a Stathmin-1a,b Ubiquitin carboxyl-terminal hydrolase isozyme L1a

IPI00220301.5 IPI00014808.1

5 1

26.8 3.9

d 5.3

IPI00291922.2 IPI00027462.1 IPI00399212.3 IPI00479997.4 IPI00018352.1

8 4 5 1 4

34.4 42.1 26.9 5.4 26.9

6.3 1.8 d 12.4 d

protein

a c

GO analysis (molecular function)

ATP binding, deoxycytidine kinase activity, phosphotransferase activity, alcohol group as acceptor ATP binding, RNA binding, calcium ion binding, low-density lipoprotein receptor binding, unfolded protein binding, virion binding actin filament binding, cell adhesion molecule binding actin binding, fructose binding, fructose-bisphosphate aldolase activity, fructose-bisphosphate aldolase activity, tubulin binding identical protein binding L-lactate dehydrogenase activity, protein binding histone binding, phosphoprotein phosphatase inhibitor activity, protein phosphatase type 2A regulator activity peroxiredoxin activity, phospholipase A2 activity 1-alkyl-2-acetylglycerophosphocholine esterase activity, protein binding protein binding, threonine-type endopeptidase activity calcium ion binding, protein binding, signal transducer activity GTP binding signal transducer activity, tubulin binding/protein binding cysteine-type endopeptidase activity, ligase activity, omega peptidase activity, ubiquitin binding, ubiquitin thiolesterase activity

Previously shown as EBV-regulated or implicated in Burkitt’s lymphoma. b Generally detected proteins according to Petrak et al.30 and Wang et al.31 Protein spot present only in the BL41/B95.8 cell line. d Protein spot present only in the BL41 cell line. 2963



Figure 3. Verification of COMMD9, vimentin, and 14-3-3ε differential expression by Western blotting. (A) Blots of total protein extracts with specific antisera against COMMD9, vimentin, and 14-3-3ε, as indicated. GAPDH was used as internal control. (B) Anti-COMMD9 Western blots on 2D gels. The bottom panels show quantitation of the spots with PDQuest software. (C) Western blot against COMMD9 in nuclear and cytoplasmic extracts, as indicated, with c-jun and tubulin used as respective markers.

R-type 5, part of the 26S proteasome that is downregulated at the protein and functional level upon EBV infection.45 Other proteins identified in our screen appear to play a role in growth regulation and cancer formation, while some are involved in actin regulation and the cytoskeleton or metabolic processes. They include septin-11, member of a family of cytoskeletal proteins with GTPase activity, involved in cytokinesis and cellular morphogenesis;46 SET, a regulator of Nm23-H1,47 which is involved in cell migration and metastasis;48 Ran/TC4, a Ras-related nuclear protein regulating the completion of DNA synthesis;49 ribose-5-phosphate isomerase, belonging to the pentose phosphate pathway that protects against hypoxia and oxidative stress;50 glucose-6-phosphate isomerase, an enzyme involved in glycolysis;51 and deoxycytidine kinase, the main enzyme in deoxyribonucleoside salvage and also required during B and T lymphocyte development.52

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Moreover, we noticed with interest that among the novel proteins identified in our screen were three members of a recently identified family, defined by the presence of a conserved motif called the COMM (Copper Metabolism gene MURR1) domain. The family is evolutionarily conserved and consists of 10 members in vertebrates.53,54 Several studies have implicated the COMMD family as an important component of NF-kB regulation. Specifically, they inhibit NF-kB mediated transcriptional events, by controlling the ubiquitination and degradation of NFkB subunits, through their interaction with a multimeric ubiquitin ligase.5358 In our proteomic study, COMMD2, COMMD3, and COMMD9 showed reduced staining in the BL41/B95.8 cells and were therefore possibly down-regulated in response to EBV infection (Figure 2). The fact that LMP1, the transforming protein of EBV, utilizes NF-kB-activating signaling cascades, suggested to us that by down-regulating COMMD proteins, EBV could enhance the duration or strength of NF-kB activation, by reducing its ubiquitination and degradation. Antibodies are available against COMMD3 and COMMD9, but in our hands only the latter could recognize a specific band upon Western blotting. Using this antibody, we were able to confirm the proteomic result both in one-dimensional (Figure 3A) and twodimensional gels (Figure 3B). In the latter case, over 50% reduction was observed upon quantitation with the PDQuest software (Figure 3B, bottom panels), whereas for one-dimensional gels an approximate 35% reduction was seen (p < 0.002, n = 6), probably because of comigrating nonspecific bands. As shown in Figure 3C, COMMD9 appeared to be mainly cytoplasmic, with no signal detected in nuclear extracts. In order to investigate whether this result is peculiar to the specific cell lines used, we used IB4, a lymphoblastoid cell line (LCL) that was established by infection of cord blood lymphocytes with EBV. IB4 cells contain four to five copies of viral DNA and are nonpermissive to viral replication.59 As a control, we used B-lymphocytes from a healthy individual (not the same donor) . In addition, we also investigated LCL cells which were collected at 35 days post EBV infection and compared to B cells from the same donor. Figure 4 shows that, similarly to BL41/B95.8 cells, COMMD9 protein levels were also reduced in the IB4 line and in the LCL, compared to the non-infected controls. We also investigated at the protein level the possible regulation of COMMD1, since this is the best-characterized member of the family. However, we did not observe any significant alteration in its expression levels, as determined by Western blotting with a specific antibody (Figure 4). This was true for all lines tested. We also performed experiments on a different EBV-transformed BL line, BL31/B95.8. COMMD1 and COMMD9 levels were similar to those of the BL41/B95.8 cells, although due to poor growth, we could not compare it to its EBV-negative counterpart (data not shown). The presence of three COMMD family members in our proteomic screen prompted us to investigate additional members of the family. We employed semiquantitative RT-PCR, as well as real-time quantitative PCR to analyze mRNA levels, determining at the same time if regulation occurs at this level of gene expression. In all cases, normalization versus endogenous GAPDH levels was performed. Representative results with RT-PCR are depicted in Figure 5A and their quantitation in Figure 5B. In accordance with our proteomic and Western blot experiments shown above, COMMD1 expression levels did not change between the two lines, whereas COMMD2, COMMD3 and COMMD9 mRNA levels showed a statistically significant 2964



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Figure 4. Comparison of COMMD9 and COMMD1 expression between EBV-infected and non-infected cells. Western blotting was performed with specific antisera against the indicated proteins in total cell extracts from the cell lines indicated. Healthy B-cell extracts were used as control for the IB4 line. B cells used as control for the LCLs were from the same donor. GAPDH was used as internal control.

Figure 5. Quantitation of COMMD mRNA levels. (A) Representative examples of RT-PCR analysis for each COMMD family member. (B) Quantitation of RT-PCR results. Ratios of BL41/B95.8 over BL41 mRNA levels are shown (1 = no difference). (C) Real-time qPCR analysis. *p < 0.05 ; **p < 0.005. N.D. = not determined.

decrease in the EBV infected cells. Moreover, we observed reduced COMMD4, COMMD5, COMMD7, COMMD8 and COMMD10 levels, while COMMD6 showed no statistically significant difference. Similar overall results were obtained with the real-time PCR analysis (Figure 5C), with COMMD2, 3, 4, 7, and 9 showing significantly reduced levels. COMMD5 also showed reduced levels, but the statistical significance was not strong. qPCR analysis of COMMD8 and 10 gave no useful results because of poor PCR efficiency. In conclusion, our results show that upon EBV infection, 7 out of 10 members of the COMMD family are down-regulated at the mRNA level and at least three at the protein level. We are currently investigating common mechanisms that may operate in the regulation of COMMD family gene expression, as well as the effect this down-regulation has on NF-kB function and the transformed phenotype. Our data are suggestive of the hypothesis depicted schematically in Figure 6, whereby reduced levels of

COMMDs, which are negative regulators of NF-kB, may enhance the transforming signal of EBV.

’ DISCUSSION In the present study we have compared proteomic profiles between an EBV-negative Burkitt’s lymphoma cell line and the same line infected in vitro with EBV. A previous proteomic study has also used the BL41 line but compared it to a distinct, EBVpositive clone from the same patient. The latter is a lymphoblastoid cell line (LCL), which requires EBV for indefinite proliferation in cell culture, whereas BL41 and its derivatives are immortal cell lines due to accumulated mutations in p53 and other genes. Our study thus ensures that the cell lines share the shame background and EBV infection is the only difference between them. Proteomic approaches have also been employed in several studies with LCLs,15 whereas others have focused on 2965



Figure 6. Proposed model of the effect of COMMD down-regulation on B-cell transformation by EBV.

the effects of particular viral proteins, such as EBNA2,60 or proteomic changes after treatment with drugs.61 It is therefore not surprising that, despite several similar findings between the present and previous studies, there are also many differences, the most prominent being our identification of members of the COMMD family as targets for down-regulation by the virus. A small number of discrepancies were found between our results and previous studies, i.e., proteins appearing as up-regulated have been reported as down-regulated and vice versa. Rather than attribute these to differences in the cell systems used, we suggest that they may represent so-called “generally detected proteins”, recurrent, nonspecific proteins common in diverse comparative proteomic studies.30,31 Indeed, several of these proteins were also found in our comparison, and we have chosen not to discuss them, unless a robust relationship to EBV transformation was known. The effects of EBV transformation on gene expression patterns, using cDNA microarrays, have also been investigated and indeed in two of them the same cell lines as ours were used.11,13 Several similarities were found, although it is well established that in comparisons between proteomic and gene expression data in the same system, the majority of identified differences are unique to each approach. It is also clear that changes in mRNA levels do not necessarily correlate with changes in protein levels. Moreover, the sensitivity of the two technologies differs, with proteomics favoring the detection of more abundant proteins. With 2DE in particular, many of the observed changes in spot intensity may not reflect differences in protein expression but post-translational modifications, solubility effects, etc. In any case, the fact that many of the proteins identified in our study have previously been implicated in EBV transformation, through either genomic/proteomic approaches or other studies, increases the confidence for the novel findings reported here. The presence of viral proteins in the EBV-positive line was confirmed by Western blotting against LMP1 and EBNA2. However, although we included an EBV database in our search, we did not detect any viral proteins in our proteomic screen. This is probably due to the relatively low levels of EBV gene expression in BL lines, as opposed to LCLs. Nonviral proteins (CD40 and p52) that were also identified by Western blotting were again absent in our 2D gels. Other proteomic or transcriptomic studies have also failed to detect these proteins. This is not surprising given that 2D gels only separate a small fraction of the total proteins in a lysate. Prominent among the proteins in our study that were for the first time implicated in EBV transformation is the COMMD family. We were intrigued to see three members of this family as

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down-regulated in response to EBV infection. It is known that with 2DE and silver staining only a small portion of a mammalian proteome can be separated and detected. We therefore reckoned that additional members of the COMMD family may also be affected but escaped detection in our proteomic screen. We therefore investigated the entire family at the mRNA level, using RT-PCR and real-time, qPCR. Our findings suggest that at least 7 members, COMMD2, 3, 4, 7, 8, 9, and 10 are down-regulated by EBV infection, whereas COMMD1 and 6 are unaffected (COMMD5 was shown to be reduced with RT-PCR, but not with qPCR). The down-regulation of COMMD9 at the protein level was also observed in two additional cell systems, the EBVinfected IB4 cell line and LCLs at 35 days post EBV infection. While additional lines as well as EBV-related tumors would have to be tested, these data suggest that our results are not a peculiarity of the BL41 line. The defining feature of the COMMD family is a unique conserved motif, the COMM domain, which is located toward the carboxyl terminus, is lysine-rich and contains conserved tryptophan and proline residues. The main role of this domain is to facilitate proteinprotein interactions. The first identified member of the family, COMMD1 is widely expressed and can play diverse roles in the cell. It was first implicated in copper metabolism, facilitating copper excretion from the liver.62 It has also been shown to interact with HIF-1R,63 probably promoting its ubiquitination and degradation. Similarly, it regulates NF-kB by associating in the nucleus with E3 ubiquitin ligase, thus stabilizing the binding between SOCS1 and the amino-terminus of RelA.53,58 The other members of the family are less well characterized; however, they all seem to share the ability to interact with NF-kB subunits and, by implication, affect their ubiquitination.53 In particular, COMMD3 and 9 have been found to bind and regulate RelB and p105. Because of limitations in the ability of BL41 cells to be transfected with genes or siRNAs, it did not prove possible to directly demonstrate a regulation of NF-kB activity by COMMDs in the EBV context. Therefore, we cannot at this point speculate about the relevance of our results for NF-k B regulation. We are currently focusing on cell types in which the critical regulators for B-lymphocyte conversion, LMP1 and EBNA2 can be expressed in isolation and which allow the necessary genetic manipulations in order to address the mechanisms involved in the attenuation of COMMD levels by EBV and the role of this regulation in B cell transformation.

’ CONCLUSION Our finding that EBV down-regulates members of the COMMD family of proteins in BL41 cells suggests a possible functional role for this regulation in oncogenic signaling by EBV in B-lymphocyte transformation, provided that these results are validated in additional cell models. One of the roles of COMMD proteins is negative regulation of NF-kB function, mediating ubiquitination and degradation of this transcription factor. Thus, a testable hypothesis can be proposed that by down-regulating COMMD proteins the virus is further enhancing its effect on NFkB activation, by attenuating its degradation. It remains to be investigated how EBV reduces COMMD expression levels and how important this regulatory mechanism is for the development of Burkitt’s lymphoma and other EBV-associated diseases. For this, it will be important to confirm our results in clinical samples of EBV-positive and negative aggressive lymphomas. 2966



’ ASSOCIATED CONTENT

bS

Supporting Information Details of peptides identified for each up-regulated (Table S1a) and down-regulated (Table S2a) protein. For each peptide, sequence, protonated mass, DeltaM, charge, probability value, Xcorr, DeltaCn, and number of ions are listed. MS/MS fragmentation data and spectra for all cases where only one peptide was detected are shown in Tables S1b and S2b. For each identified protein, molecular mass, pI, percent coverage and total peptide hits are also given. Primers used in PCR reactions are listed in Table S3. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ30 210 9655054. Fax: þ30 210 9653934. E-mail: panayotou@fleming.gr.

’ ACKNOWLEDGMENT This work was supported by a grant from the Operational Program “Competitiveness”, General Secretariat of Research and Technology, Greece (03ED422 PENED). We thank Dr. George Mosialos for his support, gift of materials, and helpful suggestions; Dr. Martina Samiotaki for help with MS analysis; Dr. M. Luftig, Dr. K. Stamatopoulos and Dr. Rowe Martin for B cells and cell lines; and Dr. S. Efthimiopoulos and Dr. P. Papazafiri for helpful comments on this work. ’ REFERENCES (1) Thorley-Lawson, D. A.; Allday, M. J. The curious case of the tumour virus: 50 years of Burkitt’s lymphoma. Nat. Rev. Microbiol. 2008, 6 (12), 913–24. (2) Young, L. S.; Dawson, C. W.; Eliopoulos, A. G. The expression and function of EpsteinBarr virus encoded latent genes. Mol. Pathol. 2000, 53 (5), 238–47. (3) Thorley-Lawson, D. A. EBV the prototypical human tumor virus--just how bad is it? J Allergy Clin. Immunol. 2005, 116 (2), 251–61; quiz 262. (4) Mosialos, G.; Birkenbach, M.; Yalamanchili, R.; VanArsdale, T.; Ware, C.; Kieff, E. The EpsteinBarr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 1995, 80 (3), 389–99. (5) Eliopoulos, A. G.; Caamano, J. H.; Flavell, J.; Reynolds, G. M.; Murray, P. G.; Poyet, J. L.; Young, L. S. EpsteinBarr virus-encoded latent infection membrane protein 1 regulates the processing of p100 NF-kappaB2 to p52 via an IKKgamma/NEMO-independent signalling pathway. Oncogene 2003, 22 (48), 7557–69. (6) Kaye, K. M.; Izumi, K. M.; Kieff, E. EpsteinBarr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (19), 9150–4. (7) Kilger, E.; Kieser, A.; Baumann, M.; Hammerschmidt, W. EpsteinBarr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J. 1998, 17 (6), 1700–9. (8) Soni, V.; Cahir-McFarland, E.; Kieff, E. LMP1 TRAFficking activates growth and survival pathways. Adv. Exp. Med. Biol. 2007, 597, 173–87. (9) Rowe, M.; Rooney, C. M.; Edwards, C. F.; Lenoir, G. M.; Rickinson, A. B. EpsteinBarr virus status and tumour cell phenotype in sporadic Burkitt’s lymphoma. Int. J. Cancer 1986, 37 (3), 367–73. (10) Calender, A.; Billaud, M.; Aubry, J. P.; Banchereau, J.; Vuillaume, M.; Lenoir, G. M. EpsteinBarr virus (EBV) induces expression of B-cell

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Comparative Proteomic Analysis Implicates COMMD Proteins as

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