Scaled-Down Purification Protocol To Access Proteomic Analysis of

May 30, 2008 - Centre d'Immunologie Pierre Fabre, 5 avenue Napoléon III, BP 497, 74164 Saint Julien en Genevois Cedex, France. Received January 30 ...
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Scaled-Down Purification Protocol To Access Proteomic Analysis of 20S Proteasome from Human Tissue Samples: Comparison of Normal and Tumor Colorectal Cells Manuelle Ducoux-Petit,‡,§,† Sandrine Uttenweiler-Joseph,*,‡,§,† Franck Brichory,| Marie-Pierre Bousquet-Dubouch,‡,§ Odile Burlet-Schiltz,‡,§ Jean-Franc¸ois Haeuw,| and Bernard Monsarrat‡,§ Universite´ de Toulouse, Institute of Pharmacology and Structural Biology, IPBS, UPS, 205 route de Narbonne, 31077, Toulouse, cedex 4, France, CNRS, IPBS, 205 route de Narbonne, 31077, Toulouse, cedex 4, France, and Centre d’Immunologie Pierre Fabre, 5 avenue Napole´on III, BP 497, 74164 Saint Julien en Genevois Cedex, France Received January 30, 2008

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses. Keywords: Proteasome purification • clinical samples • colorectal cancer • 2D gel electrophoresis • mass spectrometry • HT-29 and Caco-2 cell lines

1. Introduction Targeted protein degradation through the ubiquitin-proteasome pathway is an essential cellular process that contributes to the regulation of many cellular mechanisms including cell cycle progression, cell differentiation, signal transduction, stress responses, apoptosis and protein quality control. The multienzymatic 26S proteasome complex, present in the cytoplasm and nucleus of all eukaryotic cells, is central to this process.1 This pathway is also involved in the immune response of superior eukaryotes as some peptides generated by the proteasome are presented at the cell surface through the major histocompatibility complex (MHC) class I molecules.2 The eukaryotic 20S proteasome is the catalytic core of the 26S proteasome and is arranged in 4 stacked rings with 7 unique R subunits in the two outer rings and 7 unique β subunits in the two inner rings (Figure 1). The R subunits participate in proteasome assembly, control substrate entry, * To whom correspondence should be addressed: Dr. Sandrine Uttenweiler-Joseph, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse, France. Tel: 00 33 5 61 17 54 72. Fax: 00 33 5 61 17 59 94. E-mail: [email protected]. ‡ Universite´ de Toulouse. § CNRS, IPBS. † These authors equally contributed to this study. | Centre d’Immunologie Pierre Fabre.

2852 Journal of Proteome Research 2008, 7, 2852–2859 Published on Web 05/30/2008

and associate to regulatory particles such as PA28 and PA700. The β subunits contain three distinct active sites responsible for proteolytic activities. The β1, β2 and β5 subunits show peptidylglutamyl peptide hydrolyzing, trypsin-like and chymotrypsin-like activities, respectively. In higher eukaryotes, the subunit composition of this standard proteasome can vary in response to stimuli. For example, induction of immune response by interferon-γ (IFNγ) causes the constitutive catalytic subunits β1, β2 and β5 to be replaced by the immunosubunits, β1i, β2i and β5i, respectively.3 This newly formed complex is called immunoproteasome. The proteasome subunit composition can be even more complex because of the existence of multiple subunit isoforms, including various post-translational modifications, as shown in mammalian 20S proteasome.4,5 Each cell line or tissue may constitutively contain varying proportions of standard 20S proteasome and immunoproteasome.6 This ratio can also vary according to environmental factors, like the presence of cytokines,3 the cell differentiation state,7 or aging.8 A change in 20S proteasome composition alters the overall proteasome proteolytic activity because the different proteasome complexes (like standard proteasome and immunoproteasome) exhibit different catalytic efficiencies.9,10 From a functional point of view, these catalytic variations have a well-defined impact on the processing of MHC class I 10.1021/pr8000749 CCC: $40.75

 2008 American Chemical Society

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Scaled-Down 20S Proteasome Purification Protocol

Table 1. Clinico-Pathological Data for the Colorectal Cancer Cases under Study patient no.

sex/age in years

cancer stagea

anatomical location

1 2 3 4 5 6 7 8

M/57 M/77 M/88 M/53 M/73 M/92 F/55 M/73

pT3N0M0 pT3N1M0 pT3N0M0 pT3N0M0 pT3N0M0 pT3N1Mx pT3N0M0 pT3N0Mx

Cecum Right colon Rectum Sigmoid Left colon Left colon Right colon Sigmoid

a Tumor staging according to the TNM system; Mx: metastatic status not assessable.

work with the small number of cells available from these samples, we scaled-down and optimized the proteasome purification protocol that remained compatible with proteomic analyses. We have then been able to compare the subunit composition and activity of the 20S proteasome from paired samples of tumor and neighboring normal colorectal epithelial cells from eight patients with colorectal cancer.

2. Experimental Procedures

Figure 1. Comparison of the initial and scaled-down 20S proteasome immuno-purification procedures.

antigenic peptides,11 but their consequences for other biological processes have not yet been well-described. Therefore, the study of the structure/activity relationship of human 20S proteasome has a main interest in numerous pathologies where the proteasome is likely involved, like several neurodegenerative disorders or cancer.12,13 Furthermore, the proteasome represents a promising pharmaceutical target in antitumor therapy since proteasome inhibitors have been shown to selectively induce apoptosis in tumor cells and the proteasome inhibitor bortezomib has been approved for clinical use in the treatment of multiple myeloma.14 Moreover, only little information is currently available about the differential composition and activity of the human proteasome between normal and tumor cells. Thus, determination of the 20S proteasome status in tumor cells could be very informative. Purification of mammalian proteasome from representative cancerous cell lines can be performed using different procedures involving either chromatographic steps or immunoaffinity techniques.15–17 20S proteasome subunit composition can then be efficiently determined by 2DE subunit separation combined to mass spectrometry identification.4,17 In parallel, the proteolytic activity of purified 20S proteasome can be assessed by measuring the 3 main catalytic activities with fluorogenic substrates.18 In this study, we investigated the proteasome status in normal and tumor colorectal cells. Colorectal cancer is one of the most common cancers worldwide and is the second leading cause of cancer death in Western countries.19 Despite the introduction of new therapies combining several antitumor drugs, the survival rates among patients with advanced colorectal cancer remain poor.20 We first purified the 20S proteasome from colorectal cell lines and analyzed their subunit composition by 2DE and mass spectrometry using a previously described procedure.17 We then analyzed the proteasome status in epithelial colorectal cells from tissue samples. To be able to

2.1. Cell Culture. The HL60 promyeloblast cell line and the HT-29 and Caco-2 colorectal adenocarcinoma cell lines were obtained from the ATCC. The human cell lines HL60, HT-29 and Caco-2 were cultured in RPMI 1640, McCoy 5a and DMEM medium, respectively, supplemented with 10% fetal calf serum (20% for Caco-2), 2 mM glutamine and antibiotics (all from Invitrogen, Cergy-Pontoise, France). The cells were maintained in a humidified incubator at 37 °C in the presence of 5% CO2. 2.2. Patients and Samples. Paired samples of normal and tumor colorectal tissue derived from eight patients with colorectal cancer were obtained after surgical resections performed in various hospitals in France, according to specific agreements with hospitals and physicians (study protocol N°F50047-001). Patients were informed by physicians about the aim and scope of the research project and provided informed written consent to the study. Authorizations to collect, store and use personal genetic and medical data were obtained from the French Ministry of Research and the French Commission Nationale de l’Informatique et des Liberte´s. The collected data were made anonymous. Patients who had received any chemoand/or radio-therapeutic treatment before surgery were excluded from this study. Selected clinico-pathological data for the patients included in the study are presented in Table 1. Tissue samples were collected from various sites in the colon and rectum. Tumors were staged according to the TNM classification.21 All adenocarcinomas were at stage T3, with tumor invading through the muscularis propria. The patients did not present any distant metastasis (M0), but two patients presented pericolic lymph node involvement (N1). Normal tissue samples were taken upstream at a distance of 10-20 cm from the tumor, depending on the tumor localization.22 Tissue samples were checked and prepared by a pathologist immediately after resection. Especially, normal tissue samples were collected after pathological examination to confirm absence of polyps, adenomas and others premalignant lesions. After the pathologist control, normal and tumor tissue samples were washed and maintained at 2-8 °C in RPMI 1640 medium (Cambrex, Emerainville, France) supplemented with 100 units/ mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B (Cambrex) and protease inhibitors (EDTA-free ComJournal of Proteome Research • Vol. 7, No. 7, 2008 2853

research articles plete, Roche, Meylan, France). They were maintained in these conditions during the transport to the Centre d’Immunologie Pierre Fabre to be further treated. 2.3. Epithelial Cell Isolation. Epithelial cells were isolated from normal and tumor samples received within 1 to 2 h after surgical removal, except for samples from patient no. 7 which were received from the Hoˆpital Europe´en Georges Pompidou in Paris and treated 24 h after resection. For normal samples, the mucosa was first dissected from the submucosa and incubated for 15 min at room temperature in RPMI 1640 medium with 5 mM DTT. It was then incubated twice at 4 °C with 10 mM DTT, and remaining fragments of sublayers and blood vessels were removed with scalpel and scissors. The dissected normal mucosa and the tumor sample were cut into small pieces and incubated for 3 h at 37 °C under gentle stirring in RPMI 1640 medium containing the following enzymes to dissociate epithelial cells: collagenase A (1 mg/mL), dispase II (2 mg/mL) and Dnase I (40 µg/mL). The cell suspension was filtered through a 850 µm grid, then through a 100 µm filter. Cells were collected by centrifugation at 400g for 10 min at 4 °C, suspended in PBS and counted. The viability of the isolated epithelial cells was determined using the trypan blue method and was found to be 96.4 ( 4.3 and 92.6 ( 5.1% for normal and tumor cells, respectively. The average number of cells obtained per patient was around 138 × 106 cells from the tumor tissues and 382 × 106 from the normal tissues. Cells were characterized by flow cytometry with a FITC-labeled mouse anti-human EpCAM (CD326) antibody (clone HEA-125, Miltenyi Biotech, Paris, France). The percentage of EpCAM-positive cells was estimated by analysis of cell surface staining using a FACScan cytometer (BD Biosciences, Le Pont de Claix, France), and was determined to be 45.3 ( 12% for normal and 55 ( 12.3% for tumor cells. Isolated cells were washed three times with PBS and stored as pellets at -80 °C. 2.4. 20S Proteasome Immuno-Purification. 2.4.1. Initial Protocol. The 20S proteasome was purified by immuno-affinity chromatography from more than 1 × 109 cells as previously described.17 Briefly, the cell pellet was resuspended in 40 mL of 20 mM Tris-HCl, 50 mM NaCl, 10 mM EDTA, pH 7.6, protease inhibitors (EDTA-free Complete, Roche), and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and 20 mM sodium fluoride). Cells were broken by three consecutive freeze/thaw cycles (liquid nitrogen/30 °C water bath) and the lysate was centrifuged at 48 000g for 2 h at 4 °C to get the cytoplasmic fraction. This fraction was incubated with sepharose beads covalently coated with the monoclonal antibody MCP21 (IgG1; ECACC) directed against the human R2 subunit. The beads were packed on a column further connected to a FPLC system and the proteasome was eluted with 2 M NaCl. The concentration of proteasome was evaluated by measuring the absorbance of the eluate at 280 nm. The eluate was then dialyzed against H2O to remove the salt before 2DE analysis (dialysis membrane with a cutoff at 12 kDa). 2.4.2. Scaled-Down Protocol. For the colorectal samples and the HL60 cells, cell pellets (from 25 to 265 × 106 cells) were resuspended in 10 mM Tris-HCl, pH 7.5, and EDTA-free protease inhibitor (Roche) at the ratio of 1 mL/25 × 106 cells. Cells were broken by three consecutive freeze/thaw cycles (liquid nitrogen/30 °C water bath). The sample was homogenized in a 2 mL Dounce and sonicated. Several centrifugations of the supernatants were made: 1000g for 15 min at 4 °C, then 15 000g for 10 min at 4 °C and finally 105 000g for 1 h at 4 °C. 2854

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Ducoux-Petit et al. This final supernatant was cytoplasmic fraction. The concentration of protein in the extract was determined by the Bradford method using BSA as standard. The cytoplasmic proteasome was then purified by affinity chromatography with the monoclonal antibody MCP21 coupled with magnetic beads prepared as follows. M-280 Tosylactivated Dynabeads (20 × 109 magnetic beads; Dynal Biotech ASA, Norway) were washed twice in 10 mL of 0.1 M borate, pH 9.5, for 2 min at room temperature. Slow tilt rotation was used for all incubations and washes. For the coupling reaction, 8 µg of MCP21 antibodies was incubated in 0.1 M borate, pH 9.5, for 24 h at 37 °C with 107 beads at the concentration of 2 × 109 beads/mL. The coated beads were then washed twice in PBS and 0.2 M glycine for 5 min at 4 °C; once in 0.2 M Tris-HCl, pH 8.5, and 0.2 M glycine for 4 h at 37 °C; once in PBS and 0.2 M glycine for 5 min at 4 °C; once in 0.5% Tween-20 for 10 min at room temperature; twice in 0.1 M citrate, pH 3.1, for 5 min at room temperature and finally twice in PBS for 5 min at room temperature. The cytoplasmic fraction was incubated with the MCP21coated magnetic beads (1 × 109 beads/mL for 1 h at 4 °C). The beads were washed three times with PBS, and proteasome was then eluted with 0.1 M citrate, pH 3.1 (75 µL for 1 × 109 beads) and the eluate was immediately neutralized with 2 M Tris buffer, pH 8. 2.5. Chymotrypsin-like Activity Assay of Proteasome. This assay was performed in 96-well black plates (Greiner Bio-One, Frickenhausen, Germany) with a protocol adapted from Tenzer & Schild.23 Fifty microliters of cytoplasmic fraction from colorectal samples was added to the same volume of Suc-LLVYAMC substrate (BIOMOL International LP, Plymouth Meeting, PA) at a final concentration of 200 µM/well, and lactacystin (BIOMOL International LP) was added at 10 µM/well when necessary; lactacystin is a specific proteasome inhibitor, and at 10 µM, it totally inhibits the chymotrypsin-like activity of proteasome. The chymotrypsin-like activity specific to the proteasome can thus be calculated as the activity in absence of lactacystin minus the activity in its presence. The kinetic assays were performed at 37 °C in a FLX-800 spectrofluorimeter (BIO-TEK, Winooski, VT) over 90 min with one reading every 5 min, at 360 nm for excitation and 460 nm for emission. 2.6. 2DE separation and Image Analysis. 20S proteasome subunits were separated using pH 3-10 non-linear IPG strips in the first dimension followed by 12.5% SDS gel in the second dimension, exactly as previously described.24 Proteins were detected by Coomassie Blue staining or silver nitrate staining.17 Gels were scanned with the GS-800 densitometer (Bio-Rad) and analyzed with the ImageMaster 2-D Platinum V6.01 software (GE Healthcare). 2.7. Protein Identification. 2.7.1. In-Gel Protein Digestion. Proteins were digested with trypsin and tryptic peptides were extracted from the gel pieces as previously described.17 2.7.2. MALDI-TOF MS Analysis. MALDI-TOF MS analyses were performed on a MALDI-TOF/TOF instrument (4700 Proteomics Analyzer; Applied Biosystems, Foster City, CA). A total of 0.5 µL of tryptic digest supernatant was loaded onto the MALDI target plate and air-dried; 0.3 µL of matrix solution [R-cyano-4-hydroxycinnamic acid; 5 mg/mL in 50% acetonitrile (ACN), 0.1% trifluoroacetic acid] was then added. Mass spectra were acquired in an automated positive reflector mode from m/z 700 to 3500. Trypsin autolytic peptides (m/z 842.5100 and 2211.1046) were used to internally calibrate each spectrum to a mass accuracy within 50 ppm. Spectra were analyzed using

Scaled-Down 20S Proteasome Purification Protocol GPS Explorer (version 3.6, Applied Biosystems) which acts as an interface between the Oracle database containing raw spectra and the Mascot search engine (version 2.1.04; Matrix Science, London, U.K.). Peptide peaks with a signal/noise ratio greater than 10 were searched against human sequences in the Swiss-Prot database (Release 52.0). Two missed cleavages were allowed and the data were searched using carbamidomethylation of cysteine as a fixed modification and oxidation of methionine and N-acetylation of the proteins as variable modifications. A protein was considered correctly identified if the “protein score confidence interval” calculated by the GPS Explorer Results Browser was greater than 95%. 2.7.3. NanoLC-ESI-Q-TOF MS/MS Analysis. When MALDITOF MS data were not conclusive, protein identification was confirmed by nanoLC-ESI-MS/MS analyses. Tryptic peptide extracts were subjected to nanoLC-MS/MS analysis on an ESIQ-TOF mass spectrometer (QSTAR XL, Applied Biosystems) operating in positive mode with a 2.1 kV spray voltage. Chromatographic separation was performed on a 75 µm i.d. × 15 cm PepMap C18 column (Dionex/LC Packings, Sunnyvale, CA) at a flow rate of 200 nL/min using an increasing linear gradient of ACN in water (5-50%) over 40 min with 0.1% formic acid. Data were acquired with Analyst QS (version 1.1, Applied Biosystems). MS spectra were acquired for 1 s. For each MS spectrum, the two most intense multiple charged peaks were selected for generation of subsequent CID mass spectra. The CID collision energy was automatically adjusted according to peptide charge and m/z. A dynamic exclusion window was applied within 30 s. Data were analyzed using Analyst QS software (version 1.1) and MS/MS centroid peak lists were generated using the Mascot.dll script (version 1.6b21). Data were searched against human sequences in the Swiss-Prot database (Release 52.1) using Mascot (version 2.1.04). Peptide tolerance in MS and MS/MS modes was 0.5 Da. The other search parameters (number of missed cleavages, fixed and variable modifications) were identical to those used for peptide mass fingerprinting. Identification was considered positive if the protein was identified either with at least two peptides with a score greater than the significance threshold score of 30 (p < 0.05) or with one peptide with a score greater than the significance threshold score of 37 (p < 0.01), as determined by the Mascot Search program.

3. Results and Discussion 3.1. Proteomic Analysis of the 20S Proteasome from Human Colon Carcinoma Cell Lines. The 20S proteasomes from two human colon carcinoma cell lines, HT-29 and Caco2, were isolated from more than 1 × 109 cells using the immuno-purification method currently used in our laboratory.17 This protocol will be referred to as the initial 20S proteasome immuno-purification procedure (Figure 1). The 20S proteasome subunits were then separated by 2DE and identified by mass spectrometry (Figure 2 and Supporting Information). These 20S proteasome 2DE maps are the first ones obtained for colorectal carcinoma cell lines. They showed that the Caco-2 and HT-29 20S proteasome fractions were almost pure with few contaminants. The two 2DE maps are similar and demonstrate that both cell lines only contain standard proteasome, as described for human erythrocytes.4 However, this contrasts with other types of human cancer cell lines, including promyelocytic leukemia cell line HL60 and leukemic monocyte lymphoma U937, in which both standard proteasome and immunoproteasome were found.17,24 These results

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Figure 2. 2DE maps of 20S proteasome purified from Caco-2 (A) and HT-29 (B) colon cell lines. The 20S proteasome from cytoplasmic extracts of around 1 × 109 Caco-2 and HT-29 cells was purified by immuno-affinity using MCP21-coated sepharose beads. The proteasome subunits were then separated by 2DE using a pH 3-10 non-linear IPG strip in the first dimension followed by a 12.5% SDS gel in second. The 2D gels were stained with Coomassie blue. All labeled spots were identified by mass spectrometry (see Supporting Information).

obtained at the protein level also differ from the ones obtained by RT-PCR for other colon cancer cell lines (HCT116, SW403 and LoVo) which revealed the presence of mRNA for the immunoproteasome subunits.25 However, these various findings are not necessarily conflicting because each cell line or tissue may constitutively contain different proportions of standard 20S proteasome and immunoproteasome,6 and this ratio can also vary according to environmental factors.3,7,8 The change in 20S proteasome composition alters its overall proteolytic activity and the functional consequences of these variations are unclear, apart from MHC class I antigenic peptide processing.11 It is not well-known if 20S proteasome activity and subunit composition are modulated in cells during carcinogenesis. To explore this issue, our aim was to compare the 20S proteasomes from normal and tumor colorectal epithelial cells purified from tissues collected from patients with colorectal cancer. However, the number of cells collected from each patient could be very small (around 20 to 50 × 106 cells) and the initial protocol we used for proteasome immunopurification was limited to quantities above 300 × 106 cells (data not shown). Therefore, we first developed a new protocol to scale-down proteasome purification from mammalian cells. 3.2. Scale-Down and Optimization of the 20S Proteasome Immuno-Purification Using Magnetic Beads. We chose to use the immuno-purification method with the MCP21 antibody (directed against the human R2 subunit of 20S proteasome) because this purification procedure requires only one step to generate high purity 20S proteasome samples. MCP21 antibodies were coated on magnetic rather than sepharose beads to facilitate batch mode throughout the purification procedure (Figure 1). Thus, material loss during transfer was minimized and dead volumes were eliminated. The salt elution step was replaced by a pH step to avoid the dialysis otherwise necessary for 2DE. The scaled-down protocol was optimized as concerns Journal of Proteome Research • Vol. 7, No. 7, 2008 2855

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Figure 3. Comparison of 2DE maps of 20S proteasome immunopurified from HL60 cells by the initial (A) and the scaled-down (B) procedures. The 2DE maps of HL60 20S proteasome were obtained after purification by immuno-affinity (A) from 1200 × 106 cells with the MCP21 antibody coupled with sepharose beads and (B) from 30 × 106 cells with MCP21 coupled with magnetic beads. After Coomassie blue staining of the 2DE gel (A), the proteasome subunits were identified by mass spectrometry. The 20S proteasome 2DE map obtained with the scaled-down procedure (B) was visualized by silver staining.

(i) the chemical surface functionality for the direct coupling of the MCP21 antibodies, (ii) the coupling buffer, (iii) the quantity of antibodies per quantity of magnetic beads, (iv) the quantity of coupled magnetic beads per volume of cytosolic extract, (v) the washing conditions before elution, and (vi) the volume and the type of buffer for elution (data not shown). The HL60 cell line was chosen to compare the efficiency of the two protocols: we established in preliminary experiments that these human promyelocytic leukemia cells possess a mixture of standard and immuno 20S proteasomes (unpublished results). Thus, this cell line is a good model for following the purification of both types of proteasome. Figure 3 presents the 2DE maps obtained after immuno-purification of the proteasome from 1200 × 106 HL60 cells by the initial procedure (Figure 3A) and from 30 × 106 cells by the scaled-down protocol (Figure 3B). The 2DE gel obtained with the larger number of cells was used to identify the proteasome subunits by mass spectrometry analyses (Figure 3A and Supporting Information). The 2DE maps were very similar and revealed a mixture of immunoproteasome and standard proteasome; the only difference detected was the presence of few high Mr contaminants in the fraction obtained with the scaled-down protocol. This comparative experiment validated the protocol developed to purify proteasomes from a small quantity of human cells. The smallest number of cells necessary for the scaled-down procedure was estimated to be 2856

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Figure 4. Comparison of 2DE maps of 20S proteasomes purified from normal (A) and tumor (B) colorectal cells from patient no. 2. The 20S proteasome from the cytoplasmic extract of 50 × 106 normal colorectal cells (A) and 90 × 106 tumor cells (B) from the same patient was purified by the scaled-down immuno-affinity protocol. The proteasome subunits were then separated on 2DE gels and stained with silver nitrate. All labeled spots were identified by mass spectrometry (see Supporting Information). IgG Fc BP, IgG Fc Binding Protein; GAPDH, glyceraldehyde-3phosphate dehydrogenase; Hb, Hemoglobin.

25 × 106 cells. Therefore, this protocol was used to purify and characterize proteasomes from human biopsy samples. 3.3. Subunit Composition and Activity Measurements of 20S Proteasome Purified from a Series of Normal and Tumor Colorectal Tissue Samples. The scaled-down protocol was used to purify the cytoplasmic 20S proteasome of epithelial cells enriched from normal and tumor colon tissue samples collected from eight patients (see Table 1 and Experimental Procedures). The purified proteins were separated by 2DE and stained with silver nitrate. The 2DE gels obtained from 50 × 106 normal cells and 90 × 106 tumor cells from patient no. 2 are presented as representative gels in panels A and B of Figure 4, respectively. These 2DE maps are similar to those generally observed for the human 20S proteasome, with some additional spots at high and low Mr. MS analyses of all the spots detected confirmed the presence of 20S proteasome subunits and identified three additional proteins: hemoglobin (Hb), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and IgG Fc binding protein (IgG Fc BP). The presence of these proteins,

Scaled-Down 20S Proteasome Purification Protocol which may be associated with the proteasome or be contaminants, can be explained. The GAPDH protein has been found to co-purify with proteasome in various species and following various affinity-based purification procedures.26,27 This protein may interact with the proteasome28 or even be a proteasome substrate; indeed, its degradation by the proteasome has been demonstrated in vitro.29 The IgG Fc BP identified is mainly expressed in placenta and colonic epithelial cells30 and its presence may be a consequence of its interaction with the MCP21 IgG antibody used for the immuno-purification. The hemoglobin found is likely coming from red blood cells that remained in the enriched epithelial cell fraction. As expected, only 20S proteasome subunits were identified by mass spectrometry analyses in the boxed regions (insets of Figure 4). All the proteasome subunits were identified, except the β1 and β5i subunits. Both the tumor and normal colorectal samples contained catalytic subunits from the standard (β2 and β5) and immunoproteasome (β1i and β2i), so both contained standard and immuno 20S proteasomes. Out of the eight patients, six paired 2DE maps gave interpretable results (patients no. 1, 2, 3, 4, 6 and 7, Table 1) and all of them were comparable to those shown in Figure 4; all revealed the presence of a mixture of standard and immuno-proteasomes (data not shown). The 20S proteasome population present in the epithelial cells from colon tissue samples differed from that observed in the colorectal cell lines Caco-2 and HT-29 in which only standard proteasome was detected. Several hypotheses could explain the differences observed. The presence of non-epithelial cells cannot be excluded and these cells may contain immunoproteasome. There are also other more physiological possibilities. Although a cell type can have similar functional properties in vitro and in vivo, the growth in culture can lead to phenotypic changes that result in alterations of protein expression. To our knowledge, no large-scale proteomic analysis has been performed to compare protein expression in colon epithelial cells in vivo and in colorectal cell lines. However, a study of this type has been reported for endothelial cells and the results showed a large difference, with 41% of luminal endothelial cell plasma membrane proteins expressed in vivo not detected in vitro.31 The difference in 20S proteasome subunit composition between colon epithelial cells from human tissue and cell culture may also be a consequence of the microenvironment in vivo. Epidemiological studies have shown that chronic inflammation predisposes individuals to certain cancers, including colon cancer, and this cancer-associated inflammation promotes tumor growth and progression.32 This inflammatory process at the tumor site may influence the proteasome composition, because cytokines, including IFNγ, can induce the expression of immuno-subunits.3 The six pairs of interpretable 2DE maps were then subjected to image analysis to compare the subunit composition of the cytoplasmic 20S proteasomes from tumor and corresponding normal colorectal samples. No significant qualitative or quantitative difference was detected. To evaluate the proteolytic function of the 20S proteasome in these normal and tumor samples, their catalytic activities were measured with a specific fluorogenic substrate. As the quantity of purified proteasome was too low to perform both 2DE maps and enzymatic experiments, the proteasome activity was assayed in the cytoplasmic extract before purification. For this reason, only the chymotrypsin-like activity was measured as the lactacystin-sensitive cleavage of the proteasome sub-

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Figure 5. Proteasome chymotrypsin-like activity from normal and tumor colorectal cells of patients suffering from colorectal cancer. The proteasome activity was measured from the cytoplasmic extract of the enriched normal and tumor colorectal epithelial cells of five patients. The proteasome chymotrypsin-like activity was measured as lactacystin-sensitive cleavage of the proteasome substrate suc-LLVY-AMC (see Experimental Procedures) and is reported relative to the total protein amount.

strate suc-LLVY-AMC. The proteasome activity was normalized to the total protein amount because it was not possible to determine the quantity of proteasome present in each sample. Indeed, the presence of an IgG Fc-binding protein in the cytoplasmic extract and purified proteasome fraction prevented the use of the sandwich ELISA assay developed in our laboratory based on IgG1 MCP21 as the capture antibody. As the number and quantity of available samples were limited, we could only measure the proteasome chymotrypsin-like activity for five patients (pairs of normal and tumor tissues; Figure 5). The proteasome activity in all the normal colorectal epithelial cell samples was analogous, that for the tumor cells differed by up to 10-fold between patients. For patients no. 1 and 4, the proteasome activity in normal and tumor cells was quite similar. For the three other patients, the proteasome activity of tumor cells was higher. For patient no. 2, the activity was 4 times higher in the tumor cells, although the proteasome 2DE maps (Figure 4) revealed no significant difference. Thus, the difference in activity could not be explained by a difference of the 20S proteasome subunit composition. There are various possible causes of the differences between the proteasome activity in normal and tumor cells: (i) the quantity of 20S proteasome (which could not be measured in the cytoplasmic extract of these samples) that may also differ due to cellular relocalization (trafficking between the nucleus and cytoplasm); (ii) differences in the subunit composition of the 20S proteasome, but no significant change was observed in all the 2DE maps analyzed; (iii) the presence and nature of regulatory factors that modulate proteasome activities. Indeed, the 20S proteasome can associate to several types of regulatory complexes (19S, PA200, PA28Rβ, PA28γ) and also protein partners that can modify the overall proteasome activity and function in cells depending on the cellular environment. For example, the PA28γ proteasome regulator can stimulate the hydrolysis of peptides,33 and is up-regulated in colorectal tumor cells relative to adjacent normal tissue.34 Therefore, it would be of main interest to further adapt this scaled-down 20S proteasome immuno-purification procedure to the analysis of the integral proteasome complexes. To our knowledge, this is the first study that compares the proteasome activity of normal and tumor colorectal samples Journal of Proteome Research • Vol. 7, No. 7, 2008 2857

research articles from the same patients. These preliminary results show that the proteasome activity in tumor colorectal cells is similar to or higher than that in normal cells. The greatest difference in proteasome activity was observed for patient no. 2, the only patient among those tested for proteasome activity presenting pericolic lymph node involvement (N1) (Table 1). But additional samples need to be investigated to allow statistical analysis and assess correlations with cancer stage or site. Our description of the subunit composition and activity of 20S proteasome from normal and tumor cells is the first study producing such comparative data for colorectal cancer. Comparable data are, however, available for other types of tumors. In hematological malignancies, the concentrations of proteasome are consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults.35 Proteasome activity was found to be higher in myeloma cells from patients with chronic myeloid leukemia or multiple myeloma than in normal marrow cells.36 A recent study also showed that, in primary leukemia cells from acute lymphoblastic, chronic lymphocytic or acute myeloid leukemia, proteasome activity appeared to be higher in several, but not all samples, than in primary monocytes.37 In human breast cancer, proteasome activity in the cancerous cells was high and associated with high-level expression of proteasome subunits.38 Immunohistochemical analyses of human glioblastomas indicated that, in some cases, there are more catalytic immunosubunits than in normal peritumoral tissue.39 All these results suggest that the proteasome level tends to be higher in cancerous than normal cells, and that this is usually correlated with a higher proteolytic activity. However, this tendency does not seem to apply to all the patients suffering from the same disease. Our findings for the normal and tumor cells of patients with colorectal cancer conform to this pattern.

4. Conclusions The scaled-down protocol of proteasome immuno-purification we developed allowed us to establish the proteasome subunit composition of human colorectal cells with a limited number of cells (around 30 × 106 cells) from clinical tissue samples. We used proteomic analyses to establish, for the first time, the subunit composition and activity profile of 20S proteasome complexes from cancerous and corresponding healthy human colorectal cells. The clinical and functional pertinence of the proteasome state in normal and tumor colorectal cells remains to be clarified by further studies with a larger number of patients. Such analyses, applicable to other types of cancer, could help to determine the potential of the proteasome as a tumor marker or a prognostic factor for cancer treatment. Abbreviations: Hb, hemoglobin; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IgG Fc BP, IgG Fc binding protein; IFNγ, interferon-γ; MHC, major histocompatibility complex; 2DE, two-dimensional gel electrophoresis; ACN, acetonitrile.

Acknowledgment. We thank Dr. Franc¸ois Grange, Vale´rie Barbie´, Wim Vermeulen, Laurence Maroteix and Se´verine Martin, from Pierre Fabre Company, for managing collection of tissue samples and medical data. We thank also the physicians, Prof. Berger (Paris, France) and Drs. Grandcle´ment (Oyonnax, France), Al Naasan (Chambe´ry, France), Duprez (Annecy, France), Sautier (Cluses, France), Soualmi (Saint-Julien-en-Genevois, France), Khalaf (Belley, France), and the pathologists, Drs. Chalabreysse 2858

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Ducoux-Petit et al. (Lyon, France), Roux (Chambe´ry, France), Istier (Annecy, France), Me´atchi (Paris, France), Chenal (Chambe´ry, France), for their participation in the study. We are grateful to Blandine Pineau, Isabelle Fre´maux and David Dayde´ for their excellent technical assistance. This work was supported by a grant from the French Ministry of Industry (program “Apre`s Se´quenc¸age Ge´nomique: prote´omique et nouvelles cibles the´rapeutiques”, agreement N°004906051) and in part by grants from the CNRS (program ACI, Prote´omique et Ge´nie des Prote´ines), the Ge´nopole Toulouse Midi-Pyre´ne´es (program Biologie-Sante´), the Re´gion MidiPyre´ne´es and the Cance´ropole Grand Sud-Ouest.

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