Article pubs.acs.org/jpr
Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines Bertrand Fabre,# Thomas Lambour,# Luc Garrigues, Manuelle Ducoux-Petit, François Amalric, Bernard Monsarrat, Odile Burlet-Schiltz,* and Marie-Pierre Bousquet-Dubouch* CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale); 205 route de Narbonne, F-31077 Toulouse, France Université de Toulouse; UPS; IPBS; F-31077 Toulouse, France S Supporting Information *
ABSTRACT: The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly. KEYWORDS: formaldehyde cross-linking, affinity purification mass spectrometry (AP-MS), absolute quantification, assembly chaperones, 19S regulator
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INTRODUCTION The ubiquitin/proteasome system (UPS) is responsible for the degradation of most intracellular proteins.1 In this pathway, a protein substrate is tagged with a poly ubiquitin chain which induces its recognition and degradation by the proteasome.2 Its wide range of substrates gives a central role to the proteasome in regulating key cellular processes such as cell cycle progression, protein quality control, DNA repair, transcription, signal transduction or antigen presentation.3−6 Proteasomes are multicatalytic complexes composed of a core particle, the 20S proteasome, associated with regulators that stimulate its activity.2 The 20S proteasome is a 750 kDa barrelshaped particle made up of two identical outer α-rings and two identical inner β-rings. Each α- or β-ring is composed of seven α (α1 to α7) or β (β1 to β7) subunits, respectively. The proteolytic sites are buried within a central chamber in the 20S proteasome.7 There are three catalytic activities, a trypsin-like, a chymotrypsinlike, and a caspase-like attributed to the β2, β5 and β1 subunits, respectively. These subunits can be replaced by so-called immuno-subunits (β2i, β5i and β1i, respectively) to form the immunoproteasome. Recently, two intermediate 20S proteasome subtypes composed of a mixed assortment of standard and immuno catalytic subunits have been evidenced in addition to standard and immunoproteasomes.8 The α subunits’ N-terminal residues form a gate at the center of the α-ring that restricts substrate entry into the central proteasome channel and thus © 2014 American Chemical Society
sequester the proteolytic sites. Docking of proteasome regulators on the α-ring causes a conformational change of α subunits’ Ntermini which results in the opening of the gate.9 Several regulators have been characterized.2 PA28γ is a homoheptamer, and PA28αβ is a heteroheptamer composed of three PA28α and four PA28β subunits. The PA200 regulator is a 200 kDa protein exhibiting three main isoforms, only one of which is able to associate with the 20S core particle.10 For any of these regulators, the C-terminal residues of their subunits interact with pockets formed by the α-ring of the 20S proteasome.9 Even though the molecular mechanisms used by these regulators to activate proteasomes have been well characterized, the stoichiometry of their association with the 20S core particle, and their dynamics upon cell physiological changes, are far less understood.9 The 19S regulatory particle is composed of six ATPases (Rpt1−6) and 13 non-ATPases (Rpn1−3, Rpn5−13 and Rpn15) subunits.2 The 19S binds the 20S particle via an HbYX (hydrophobic residue−tyrosine−any amino acid tripeptide) motif in the C-terminus of the ATPase subunits. The ATPase subunits are also responsible for substrate unfolding and translocation into the catalytic chamber of the proteasome. The non-ATPase subunits Rpn10 and Rpn13 contain ubiquitin-binding domains (UIM and Pru, respectively) Received: February 26, 2014 Published: May 8, 2014 3027
dx.doi.org/10.1021/pr500193k | J. Proteome Res. 2014, 13, 3027−3037
Journal of Proteome Research
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and constitute the main platforms for the recognition of the poly ubiquitinated substrates.11 Thanks to the Rpn11 subunit which cleaves the bond between the substrate and the first ubiquitin, the ubiquitin chains are released from the substrate before its translocation into the core particle.2 The functions of the other non-ATPase subunits of the 19S regulator still remain poorly understood, and it is likely they are scaffolding subunits, as shown for the Rpn6 subunit.12 Several other proteins can also modulate proteasome activity, either by interacting with its regulators or directly with the 20S catalytic core.13−15 An example of interaction with the yeast 26S proteasome (a 20S capped with one 19S) is given with an E3 ubiquitin ligase, Hul5, which elongates the poly ubiquitin chains on substrates bound to the proteasome. Meanwhile the chaintrimming activity of Ubp6, a deubiquitinating enzyme, removes ubiquitin from these substrates.16 The mammalian heterologues of these enzymes are UBE3C and USP14, respectively.16 Opposite activities of proteins interacting with the 26S proteasome thus regulate the level of substrate degradation by modulating the length of ubiquitin chains. Another example of proteasome-associated proteins is given with the proteasome assembly chaperones (PACs) that bind to the 20S catalytic core. Five different chaperones have been characterized to promote the assembly of the 20S proteasome.17 The PAC3/PAC4 (proteasome assembly chaperone) heterodimer is involved in the formation of the α-ring and is released from assembly intermediates after the incorporation of the first β subunit (β2 or β2i).18 Initiation of β-ring assembly is accompanied by the binding of POMP to the assembly intermediate. POMP may help to prevent the dimerization of precursors containing incomplete sets of β subunits and is degraded upon maturation of the 20S proteasome catalytic sites after the dimerization of half-proteasomes.17 The PAC1/PAC2 heterodimer remains associated all along the proteasome assembly process and is degraded by the newly formed 20S proteasome.19,20 Both PAC1 and PAC2 contain HbYX motifs, such as the 19S and PA200 proteasomal activators.21 This indicates that PAC1/PAC2 heterodimers might prevent the binding of proteasome regulators to assembly intermediates. During the last decades, many efforts have been made to determine the structure of proteasome complexes. Crystallographic and electron microscopy studies have given many details on the organization of the 20S proteasome and its regulators.22−24 However, very few studies attempted to determine the distribution of 20S proteasome-associated regulators in the cell.25 Moreover very few data about the stoichiometry between proteasome complexes and their interacting proteins are available.11,26,27 Finally, it has been shown that the catalytic subunits composition of the 20S proteasome varies between cell types,8,28,29 but to our knowledge, few studies deal with its regulators distribution.25 Here, we used an affinity purification method and label-free quantitative proteomics to determine the composition of the 20S proteasome and the distribution of its associated regulators. This strategy has been applied to a set of nine commonly used human cell lines. Significant differences have been observed in the proportion of free 20S proteasome, in the distribution of associated regulators, and in the amount of associated proteasome assembly chaperones. These data suggest that the proteasome composition is highly adaptable and can be modulated at different levels to maintain cellular protein homeostasis.
Article
MATERIALS AND METHODS
Cell Lines and Culture Conditions
HEK 293T, HCT116, and RKO cell lines were grown in DMEM media supplemented with 10% fetal bovine serum (FBS). U937, HeLa S3, and NB4 cell lines were grown in RPMI 1640 media supplemented with 10% FBS. KG1a cell line was grown in RPMI 1640 media supplemented with 20% FBS. MRC5 cell line was grown in MEM-α media supplemented with 10% FBS. All cell lines were cultured with 2 × 10−3 M glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin at 37 °C and 5% CO2. Unsynchronized cells were harvested at 80% of confluence for adherent cells or at a concentration of 1 × 106 cells per ml of culture for suspension cells. Formaldehyde in Vivo Cross-Linking and Proteasome Purification and Quantification
Formaldehyde in vivo cross-linking was performed with a concentration of 0.1% at 37 °C during 15 min. The crosslinking reaction was quenched with addition of 125 mM of glycine, and cells were washed three times with PBS and stored at −80 °C. Cells were lysed with 2 mL of lysis buffer (10 mM Hepes pH 7.9, 10 mM KCl, 5 mM MgCl2, 10% glycerol, 10 mM ATP, 1% NP40, protease and phosphatase inhibitor (Roche)) for 15 min at 4 °C, sonicated, and centrifuged. Protein concentration was determined by detergent-compatible assay (DC assay; Bio-Rad). 20S Proteasome purification and quantification by sandwich ELISA assay were performed as previously described.30 Proteasome Activity Assay
The assay was performed in 96-well black plates (Greiner BioOne, Frickenhausen, Germany). Ten μL of each fraction lysate were added to 40 μL of Tris-HCl 100 mM and 50 μL of SucLLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like activity), and Z-LLE-AMC (for caspase activity), substrate (BIOMOL International) in 200 mMTrisHCl, pH 8 at a final concentration of 400 μM/well. The kinetic assays were performed at 37 °C in a FLX-800 spectrofluorimeter (BIOTEK, Winooski, VT, U.S.A.) over 90 min with one reading every 5 min, at 360 nm for excitation and 460 nm for emission. Each proteasome specific activity (chymotrypsin, trypsin, and caspase) was obtained by dividing the total activity measured in the lysate by the total proteasome content determined by ELISA. Detailed LC−MS/MS Analysis, Data Search and Validation
Each proteasome purification sample was precipitated with 20% TCA and washed with acetone. Samples were boiled 30 min at 95 °C in Laemmli buffer to denature proteins and reverse formaldehyde cross-link, as previously optimized.30 Proteins were alkylated with 100 mM chloroacetamide for 30 min at room temperature in the dark. Proteins were concentrated in a single band on a 12% acrylamide SDS-PAGE gel and visualized by colloidal Coomassie Blue staining. One-shot analysis of the entire mixture was performed. A single band, containing the whole sample, was cut and washed in 50 mM ammonium bicarbonate for 15 min at 37 °C followed by a second wash in 50 mM ammonium bicarbonate, acetonitrile (1:1) for 15 min at 37 °C. Trypsin (Promega) digestion was performed overnight at 37 °C. The resulting peptides were extracted from the gel by three steps: a first incubation in 50 mM ammonium bicarbonate for 15 min at 37 °C and two incubations in 10% formic acid, acetonitrile (1:1) for 15 min at 37 °C. The three collected extractions were pooled with the initial digestion supernatant, dried in a Speed-Vac, and resuspended with 2% acetonitrile, 3028
dx.doi.org/10.1021/pr500193k | J. Proteome Res. 2014, 13, 3027−3037
Journal of Proteome Research
Article
mented in the MFPaQ v4.0.0 software (http://mfpaq. sourceforge.net/).31−33 For each sample, the software uses the validated identification results and extracts ion chromatograms (XIC) of the identified peptide ions in the corresponding raw nanoLC−MS files on the basis of their experimentally measured retention time (RT) and monoisotopic m/z values. The time value used for this process is retrieved from Mascot result files, based on an MS2 event matching to the peptide ion. If several MS2 events were matched to a given peptide ion, the software checks the intensity of each corresponding precursor peak in the previous MS survey scan. The time of the MS scan which exhibits the highest precursor ion intensity is attributed to the peptide ion and then used for XIC extraction as well as for the alignment process. Peptide ions identified in all the samples to be compared were used to build a retention time matrix in order to align LC−MS runs. If some peptide ions were sequenced by MS/MS and validated only in some of the samples to be compared, their XIC signal was extracted in the nanoLC−MS raw file of the other samples using a predicted RT value calculated from this alignment matrix by a linear interpolation method. Quantification of peptide ions was performed based on calculated XIC area values. In order to perform protein relative quantification in different samples, a Protein Abundance Index (PAI) was calculated. It is defined as the average of XIC area values for the most three intense reference tryptic peptides identified for this protein (the three peptides exhibiting the highest intensities across the different samples were selected as reference peptides, and these same three peptides were used to compute the PAI of the protein in each sample; if only one or two peptides were identified and quantified in the case of lowabundant proteins, the PAI was calculated on the basis of their XIC area values).
0.05% trifluoroacetic acid. The peptide mixtures were analyzed by nano-LC−MS/MS using an Ultimate3000 system (Dionex) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Five microliters of each peptide sample corresponding to an equivalent initial quantity of 20S proteasome (estimated by Elisa) of 2.5 μg were loaded on a C18 precolumn (300-μm inner diameter × 5 mm; Dionex) at 20 μL/min in 5% acetonitrile, 0.05% trifluoroacetic acid. After 5 min of desalting, the precolumn was switched online with the analytical C18 column (75-μm inner diameter × 15 cm; PepMap C18, Dionex) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5−50% gradient of solvent B during 160 min at a 300 nL/min flow rate. The LTQOrbitrap was operated in data-dependent acquisition mode with the Xcalibur software. Survey scan MS spectra were acquired in the Orbitrap on the 350−1800 m/z range with the resolution set to a value of 60,000. The five most intense ions per survey scan were selected for CID fragmentation, and the resulting fragments were analyzed in the linear trap (LTQ). Dynamic exclusion was used within 60 s to prevent repetitive selection of the same peptide. The Mascot Daemon software (version 2.3.2; Matrix Science, London, UK) was used to perform database searches, using the Extract_msn.exe macro provided with Xcalibur (version 2.0 SR2; Thermo Fisher Scientific) to generate peaklists. The following parameters were set for creation of the peaklists: parent ions in the mass range 400−4500, no grouping of MS/MS scans, and threshold at 1000. A peaklist was created for each analyzed fraction, and individual Mascot (version 2.3.01) searches were performed for each fraction. The mass tolerances in MS and MS/MS were set to 5 ppm and 0.8 Da, respectively, and the instrument setting was specified as “ESITRAP.” Trypsin was designated as the protease (specificity set for cleavage after Lys or Arg), and up to two missed cleavages were allowed. Oxidation of methionine and amino-terminal protein acetylation were searched as variable modifications. Carbamidomethylation on cysteine was set as a fixed modification. Protein hits were automatically validated with an FDR of 5% on proteins and 1% on peptides (minimum peptide length of six amino acids). To evaluate false positive rates, all the initial database searches were performed using the “decoy” option of Mascot, i.e. the data were searched against a combined database containing the real specified protein sequences (target database, Swiss-Prot human, release 2013_01, 20232 entries) and the corresponding reversed protein sequences (decoy database). MFPaQ used the same criteria to validate decoy and target hits, calculated the false discovery rate (FDR = number of validated decoy hits/(number of validated target hits + number of validated decoy hits) × 100). Proteins identified with exactly the same set of peptides were grouped, and only one member of the protein group was reported (the one that we considered as the most significant according to the functional description given in the UniProtKnowledgebase). Highly homologous protein hits, i.e. proteins identified with top ranking MS/MS queries also assigned to another protein hit of higher score (red, nonbold peptides), were detected by the MFPaQ software31 and were considered as individual hits and included in the final list only if they were additionally assigned a specific top ranking (red and bold) peptide of score higher than 25 (p-value
