Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics
2012; Elsevier BV; Volume: 12; Issue: 3 Linguagem: Inglês
10.1074/mcp.m112.024547
ISSN1535-9484
AutoresMark Larance, Yasmeen Ahmad, Kathryn J. Kirkwood, Tony Ly, Angus I. Lamond,
Tópico(s)Peptidase Inhibition and Analysis
ResumoProtein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. Targeted protein degradation is an important regulatory mechanism that allows co-ordination of cellular pathways in response to environmental and temporal stimuli (1Ciechanover A. Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting.Cell Death Differ. 2005; 12: 1178-1190Crossref PubMed Scopus (268) Google Scholar). The control of diverse biochemical pathways, including cell cycle progression and the response to DNA damage, is mediated, at least in part, by dynamic alterations in protein degradation (2Reed S.I. Ratchets and clocks: The cell cycle, ubiquitylation and protein turnover.Nat. Rev. Mol. Cell Bio. 2003; 4: 855-864Crossref PubMed Scopus (242) Google Scholar). Previous large scale proteomics studies in mammalian cells have shown that the rate of protein degradation can vary from the timescale of minutes, to essentially infinite stability for metastable proteins (3Doherty M.K. Hammond D.E. Clague M.J. Gaskell S.J. Beynon R.J. Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.J. Proteome Res. 2009; 8: 104-112Crossref PubMed Scopus (234) Google Scholar, 4Eden E. Geva-Zatorsky N. Issaeva I. Cohen A. Dekel E. Danon T. Cohen L. Mayo A. Alon U. Proteome Half-Life Dynamics in Living Human Cells.Science. 2011; 331: 764-768Crossref PubMed Scopus (226) Google Scholar, 5Yen H.C. Xu Q. Chou D.M. Zhao Z. Elledge S.J. Global Protein Stability Profiling in Mammalian Cells.Science. 2008; 322: 918-923Crossref PubMed Scopus (326) Google Scholar, 6Schwanhäusser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. Chen W. Selbach M. Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4059) Google Scholar, 7Boisvert F.O.M. Ahmad Y. Gierlinski M. Charriere F. Lamont D. Scott M. Barton G. Lamond A.I. A quantitative spatial proteomics analysis of proteome turnover in human cells.Mol. Cell. Proteomics. 2012; 11M111.011429Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 8Savas J.N. Toyama B.H. Xu T. Yates 3rd, J.R. Hetzer M.W. Extremely long-lived nuclear pore proteins in the rat brain.Science. 2012; 335: 942Crossref PubMed Scopus (215) Google Scholar). Most intracellular proteins have similar degradation rates, with a half-life approximating the cell doubling rate. Under 5% of proteins display degradation rates more than threefold faster than the proteome average (3Doherty M.K. Hammond D.E. Clague M.J. Gaskell S.J. Beynon R.J. Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.J. Proteome Res. 2009; 8: 104-112Crossref PubMed Scopus (234) Google Scholar, 4Eden E. Geva-Zatorsky N. Issaeva I. Cohen A. Dekel E. Danon T. Cohen L. Mayo A. Alon U. Proteome Half-Life Dynamics in Living Human Cells.Science. 2011; 331: 764-768Crossref PubMed Scopus (226) Google Scholar, 5Yen H.C. Xu Q. Chou D.M. Zhao Z. Elledge S.J. Global Protein Stability Profiling in Mammalian Cells.Science. 2008; 322: 918-923Crossref PubMed Scopus (326) Google Scholar, 7Boisvert F.O.M. Ahmad Y. Gierlinski M. Charriere F. Lamont D. Scott M. Barton G. Lamond A.I. A quantitative spatial proteomics analysis of proteome turnover in human cells.Mol. Cell. Proteomics. 2012; 11M111.011429Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). However, degradation rates for individual proteins can change, for example depending on either the cell cycle stage, or signaling events, and can also vary depending on subcellular localization. Disruption of such regulated protein stability underlies the disease mechanisms responsible for forms of cancer, e.g. p53 (9Levine A.J. Momand J. Finlay C.A. The P53 Tumor suppressor gene.Nature. 1991; 351: 453-456Crossref PubMed Scopus (3653) Google Scholar, 10Vojtěsek B. Lane D.P. Regulation of P53 protein expression in human breast-cancer cell-lines.J. Cell Sci. 1993; 105: 607-612Crossref PubMed Google Scholar) and the proto-oncogene c-Myc (11Thomas L.R. Tansey W.P. Proteolytic Control of the Oncoprotein Transcription Factor Myc.Adv. Cancer Res. 2011; 110: 77-106Crossref PubMed Scopus (78) Google Scholar). Detection of rapidly degraded proteins can be difficult because of their low abundance. However, advances in mass spectrometry based proteomics have enabled in-depth quantitative analysis of cellular proteomes (12Cox J. Mann M. Quantitative, high-resolution proteomics for data-driven systems biology.Annu. Rev. Biochem. 2011; 80: 273-299Crossref PubMed Scopus (531) Google Scholar, 13Bensimon A. Heck A.J. Aebersold R. Mass spectrometry-based proteomics and network biology.Annu. Rev. Biochem. 2012; 81: 379-405Crossref PubMed Scopus (317) Google Scholar, 14Cravatt B.F. Simon G.M. Yates 3rd, J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991-1000Crossref PubMed Scopus (571) Google Scholar). Stable isotope labeling by amino acids in cell culture (SILAC) 1The abbreviations used are:APCanaphase-promoting complexBCAbicinchoninic acidCBQCA3-(4-Carboxybenzoyl)quinoline-2-carboxaldehydeCHXcycloheximideDMEMDulbecco's modified eagle mediumDNAdeoxyribonucleic acidDRiPdefective ribosomal productGOgene ontologyHESHEPES, EDTA, sucroseHRPhorseradish peroxidaseLDSlithium dodecyl sulfatemRNAmessenger RNARDPrapidly depleted proteinRTroom temperatureSCFSkp, Cullin, F-boxSECsize exclusion chromatographySILACstable isotope labeling with amino acids in cell cultureTBSTTBS tween 20TCEPtriscarboxyethylphosphineTEABtriethylamine bicarbonateUPRunfolded protein response. 1The abbreviations used are:APCanaphase-promoting complexBCAbicinchoninic acidCBQCA3-(4-Carboxybenzoyl)quinoline-2-carboxaldehydeCHXcycloheximideDMEMDulbecco's modified eagle mediumDNAdeoxyribonucleic acidDRiPdefective ribosomal productGOgene ontologyHESHEPES, EDTA, sucroseHRPhorseradish peroxidaseLDSlithium dodecyl sulfatemRNAmessenger RNARDPrapidly depleted proteinRTroom temperatureSCFSkp, Cullin, F-boxSECsize exclusion chromatographySILACstable isotope labeling with amino acids in cell cultureTBSTTBS tween 20TCEPtriscarboxyethylphosphineTEABtriethylamine bicarbonateUPRunfolded protein response. (15Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar), has been widely used to measure protein properties such as abundance, interactions, modifications, turnover, and subcellular localization under different conditions (16Lamond A.I. Uhlen M. Horning S. Makarov A. Robinson C.V. Serrano L. Hartl F.U. Baumeister W. Werenskiold A.K. Andersen J.S. Vorm O. Linial M. Aebersold R. Mann M. Advancing cell biology through proteomics in space and time (PROSPECTS).Mol. Cell Proteomics. 2012; 11O112.017731Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Subcellular fractionation and protein size separation are also powerful techniques that enhance in-depth analysis of cellular proteomes. Not only do these fractionation techniques increase total proteome coverage, they also provide biological insight regarding how protein behavior differs between subcellular compartments. For example, subcellular fractionation has highlighted differences in the rate of ribosomal protein degradation between the nucleus and cytoplasm, (7Boisvert F.O.M. Ahmad Y. Gierlinski M. Charriere F. Lamont D. Scott M. Barton G. Lamond A.I. A quantitative spatial proteomics analysis of proteome turnover in human cells.Mol. Cell. Proteomics. 2012; 11M111.011429Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 17Lam Y.W. Lamond A.I. Mann M. Andersen J.S. Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.Curr. Biol. 2007; 17: 749-760Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Other studies have also demonstrated the benefit of in-depth subcellular fractionation and created methods for the characterization of how proteomes are localized in organelles (18Gatto L. Vizcaíno J.A. Hermjako H. Huber W. Lilley K.S. Organelle proteomics experimental designs and analysis.Proteomics. 2010; 10: 3957-3969Crossref PubMed Scopus (47) Google Scholar, 19Dunkley T.P. Watson R. Griffin J.L. Dupree P. Lilley K.S. Localization of organelle proteins by isotope tagging (LOPIT).Mol. Cell Proteomics. 2004; 3: 1128-1134Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 20Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1051) Google Scholar). anaphase-promoting complex bicinchoninic acid 3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde cycloheximide Dulbecco's modified eagle medium deoxyribonucleic acid defective ribosomal product gene ontology HEPES, EDTA, sucrose horseradish peroxidase lithium dodecyl sulfate messenger RNA rapidly depleted protein room temperature Skp, Cullin, F-box size exclusion chromatography stable isotope labeling with amino acids in cell culture TBS tween 20 triscarboxyethylphosphine triethylamine bicarbonate unfolded protein response. anaphase-promoting complex bicinchoninic acid 3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde cycloheximide Dulbecco's modified eagle medium deoxyribonucleic acid defective ribosomal product gene ontology HEPES, EDTA, sucrose horseradish peroxidase lithium dodecyl sulfate messenger RNA rapidly depleted protein room temperature Skp, Cullin, F-box size exclusion chromatography stable isotope labeling with amino acids in cell culture TBS tween 20 triscarboxyethylphosphine triethylamine bicarbonate unfolded protein response. In this study we have used SILAC-based quantitative mass spectrometry combined with extensive subcellular and protein-level fractionation to identify rapidly degraded proteins in human U2OS cells. We provide a proteome level characterization of a major feedback mechanism involving inhibition of protein translation when the proteasome is inhibited. We also present the Encyclopedia of Proteome Dynamics, a user-friendly online resource providing access to the entire data set. U2OS cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco's modified Eagle medium (DMEM), fetal calf serum, antibiotics, NuPage gels, LDS sample buffer, MES SDS-PAGE running buffer, nitrocellulose iBlot stacks, SYPRO Ruby, Alexa Fluor 680-conjugated secondary antibodies, and CBQCA assay kit were obtained from Invitrogen (Carlsbad, CA). IrDye 800-conjugated secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA). HRP-conjugated secondary antibodies were from Cell Signaling Technology (Danvers, MA). bicinchoninic acid (BCA) assay reagents, Coomassie plus (Bradford) reagent, subcellular protein fractionation Kit, detergent removal plates, 16% methanol-free paraformaldehyde, Acclaim Pepmap C18 columns and trapping cartridges, and triscarboxyethylphosphine (TCEP) (Bond-breaker neutral pH solution) were from Thermo Scientific (Waltham MA). Trypsin Gold was from Promega. Sep-Pak tC18 96-well μ-elution plates were from Waters (Milford, MA). Complete protease inhibitor mixture tablets and PhosStop phosphatase inhibitor tablets were from Roche (Basel, Switzerland). All other materials were obtained from Sigma (St. Louis, MO). Briefly, U2OS cells were grown in DMEM supplemented with 10% fetal calf serum (FCS), 2 mm l-glutamine, 100 U/l penicillin, and 100 μg/l streptomycin at 37 °C in 10% CO2, and passaged at ∼80% confluence. For SILAC labeling of U2OS cells arginine and lysine free DMEM was used and supplemented with stable isotope labeled arginine and lysine in addition to dialyzed FCS as described previously (21Boulon S. Pradet-Balade B. Verheggen C. Molle D. Boireau S. Georgieva M. Azzag K. Robert M.C. Ahmad Y. Neel H. Lamond A.I. Bertrand E. HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.Mol. Cell. 2010; 39: 912-924Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). After splitting by trypsinization, cells in SILAC media were grown to ∼80% confluence over 2–3 days before drug treatment and lysis for fractionation. For the SILAC screen U2OS cells were treated with either DMSO, 40 μg/ml cycloheximide or 10 μm MG132 for 6 h and then combined in a 1:1:1 ratio of cells and fractionated by detergent solubility with the subcellular protein fractionation kit (Pierce). These fractions were then chloroform-methanol precipitated (22Wessel D. Flügge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3163) Google Scholar) and further separated by molecular weight using denaturing size exclusion chromatography before digestion and liquid chromatography-tandem MS (LC-MS/MS) analysis. Using a Dionex Ultimate 3000 HPLC system, fractions resuspended in 4% SDS, 100 mm NaCl, 25 mm TCEP, 50 mm N-ethylmaleimide, 10 mm Na PO4 pH 6.0 were heated to 65 °C for 10 min, then filtered through a 0.45 μm filter. Samples were injected (50 μl per injection - 250 μg protein) onto a mAbPacSEC column (Dionex) equilibrated with 0.2% SDS, 100 mm NaCl, 10 mm Na PO4 pH 6.0. The flow rate was 0.2 ml min−1 and 8 × 200 μl fractions were collected using a low protein binding 96-deep well plate (Eppendorf). Triethylamine bicarbonate (TEAB, 1 m pH 8.0) was added to each fraction to adjust the pH to 8.0, and trypsin diluted in 0.1 m TEAB was added at a ratio of 1:50 with incubation for 18 h at 37 °C. SDS was removed from each fraction using detergent removal resin in 96-well plates as described previously (23Bereman M.S. Egertson J.D. MacCoss M.J. Comparison between procedures using SDS for shotgun proteomic analyses of complex samples.Proteomics. 2011; 11: 2931-2935Crossref PubMed Scopus (46) Google Scholar). For peptide desalting trifluoroacetic acid (TFA) was added to 1% (v/v) final concentration and peptides were purified using a Sep-Pak tC18 96-well μ-elution plate. Peptides were eluted in 200 μl of 50% (v/v) acetonitrile 0.1% TFA and speedivaced to dryness before resuspension in 5% (v/v) formic acid. Peptide concentrations were determined using the CBQCA assay after 25-fold dilution of peptide samples in 0.1 m borate buffer pH 9.3. Using a Dionex Ultimate 3000 nanoHPLC system, 4 μg of peptides in 5% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Dionex). After washing with 2% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 50 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column over a 140 min organic gradient with a flow rate of 200 nl min−1. Peptides were ionized by nano-electrospray ionization at 1.2 kV using a fused silica emitter with an internal diameter of 5 μm (New Objective). Tandem mass spectrometry analysis was carried out on a QExactive mass spectrometer (Thermo Scientific). Data were processed, searched, and quantified using the Maxquant software package version 1.2.2.5 as described previously (24Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar, 25Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: A peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3450) Google Scholar), using the default settings and employing the Human Uniprot database (06/07/11) containing 109,824 entries. The settings used for the Maxquant analysis were: two failed cleavages were allowed; fixed modification was N-ethylmaleimide on cysteine; enzymes were Trypsin (K/R not before P); Variable modifications included in the analysis were methionine oxidation, deamidation of glutamine or asparagine, N-terminal pyro-glutamic acid formation, protein N-terminal acetylation. A mass tolerance of 7 ppm was used for precursor ions and a tolerance of 20 ppm was used for fragment ions. Using the default Maxquant settings a maximum false positive rate of 1% was allowed for both peptide and protein identification. This cutoff was used for accepting individual spectra as well as whole proteins in the Maxquant output. This threshold has previously been shown to be a rigorous method for identifying true positive matches (24Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar). For identical protein matches in both Swissprot and Tremble within the same protein group, the match in Swissprot was reported. Isoforms were recorded in the same protein group if they shared the same peptide coverage. All replicates indicated are biological replicates and protein quantitation was derived from at least two of the biological replicates. SILAC ratios for all proteins were normalized using β-Tubulin, Histone H2A, and GAPDH ratios. No outlier data points were removed. p values were calculated using a Student's t test comparing the three biological replicate SILAC ratios of each protein, to the three biological replicate SILAC ratios for the control, known stable proteins, i.e. β-tubulin, histone H2A, and GAPDH. For the CHX/DMSO (M/L) and MG132/DMSO (H/L) SILAC ratios a one-tailed and two-tailed t test was used respectively. Values were tested for normality using the Shapiro-Wilk test. For generating total cell lysates, cells were lysed in five times the total volume of cells using 2% SDS in 10 mm HEPES, 250 mm sucrose, and 1 mm EDTA (HES buffer) with 50 mm N-ethylmaleimide, Complete Protease Inhibitors EDTA-free (Roche) and PhosStop (Roche). Lysates were incubated at 65 °C and viscosity because of DNA was removed by passing the lysate through a Qiashredder (Qiagen). Equal amounts of protein were loaded for SDS-PAGE of each sample with 10 μg per lane. SDS-PAGE was performed using 4–12% (w/v) Bis-Tris NuPage gels using MES running buffer according to manufacturer's instructions but with the addition of 25 mm TCEP, in the LDS sample buffer. SYPRO Ruby staining was performed as per manufacturer's instructions (Invitrogen). For Western blotting, separated proteins were electrophoretically transferred to an iBlot nitrocellulose membrane, blocked with 3% nonfat skim milk in 0.1% Tween-20 in TBS (TBST) and incubated with primary antibody in 5% bovine serum albumin (BSA) in TBST overnight at 4 °C. After incubation, membranes were washed three times in TBST and incubated with either HRP-labeled or Alexa fluor 680/IrDye 800 labeled secondary antibodies in 3% nonfat skim milk in TBST. Proteins were visualized using Immobillon chemiluminescent substrate (Millipore) and imaged either with a cooled CCD camera (Fuji) for HRP-labeled secondary antibodies, or a Licor Odyssey CLx imager for Alexa fluor 680/IrDye 800 labeled secondary antibodies. Cells were cultured on glass coverslips as described above. All subsequent steps are at 25 °C. Cells were then fixed with 3% paraformaldehyde in PBS. Fixed cells were washed with PBS, and free aldehyde groups were quenched with 50 mm glycine in PBS. The cells were then permeabilized using 1% Triton X-100 for 10 min followed by washing in PBS. Coverslips were processed for immunolabeling by blocking with 5% BSA in TBST. Primary antibodies were incubated on coverslips for 1 h in 5% BSA in TBST. Coverslips were washed in PBS. Primary antibodies were detected with Alexa Fluor 488 or Alex Fluor 594 conjugated secondary antibodies which were incubated on coverslips for 30 min in 5% BSA in TBST with 2 μg/ml Hoechst 33342. Optical sections were analyzed by confocal microscopy on a Leica SP2 AOBS inverted microscope. Images generated are a single confocal slice from the middle of the cell monolayer. U2OS cells were transfected with 0.45 nmoles of siRNA per condition using RNAiMax (Invitrogen) in Optimem medium (Invitrogen). After 24 h, cells were harvested by trypsinization for analysis either by SDS-PAGE or flow cytometry. An asynchronous U2OS cell suspension in DMEM containing 10% FCS was incubated with 25 μg/ml Hoechst 33342 for 5 min at room temperature. Cells were sorted by DNA content into G1, S and G2-phase cell populations using an ACS Vantage DIVA instrument (BD Biosciences). Sorted cells were washed in PBS and lysed in 2% SDS in HES buffer. To identify rapidly degraded proteins human U2OS osteosarcoma cells were treated for 6 h with cycloheximide (CHX), which rapidly blocks translation elongation and hence protein synthesis. This method has been used in many studies of protein degradation (26Zhou P. Determining protein half-lives.Methods Mol. Biol. 2004; 284: 67-77PubMed Google Scholar) and is complementary to previous studies using pulse-SILAC to identify rapidly degraded proteins (3Doherty M.K. Hammond D.E. Clague M.J. Gaskell S.J. Beynon R.J. Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC.J. Proteome Res. 2009; 8: 104-112Crossref PubMed Scopus (234) Google Scholar, 6Schwanhäusser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. Chen W. Selbach M. Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4059) Google Scholar, 7Boisvert F.O.M. Ahmad Y. Gierlinski M. Charriere F. Lamont D. Scott M. Barton G. Lamond A.I. A quantitative spatial proteomics analysis of proteome turnover in human cells.Mol. Cell. Proteomics. 2012; 11M111.011429Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). To determine if rapidly degraded proteins are substrates of the ubiquitin-proteasome system a separate population of U2OS cells were treated for 6 h with the proteasome inhibitor MG132. As a control, U2OS cells were treated with DMSO for 6 h. The DMSO control, cycloheximide, and MG132 treated cells were grown, respectively, in either "light," "medium," or "heavy" SILAC media (Fig. 1A; see methods). After the 6 h incubation, the three cell populations were mixed in equal amounts and processed for MS analysis as described below (Fig. 1A). The resulting isotope-encoded proteins isolated from the mixed-cell populations were fractionated both by subcellular compartment and molecular weight (Fig. 1A). Four subcellular fractions were generated, i.e. nucleus, cytosol, cytoskeleton, and membrane. Each subcellular fraction was individually separated by protein molecular weight, using denaturing SDS size exclusion chromatography (SDS-SEC), as previously described (27Larance M. Bailly A.P. Pourkarimi E. Hay R.T. Buchanan G. Coulthurst S. Xirodimas D.P. Gartner A. Lamond A.I. Stable-isotope labeling with amino acids in nematodes.Nat. Methods. 2011; 8: 849-851Crossref PubMed Scopus (91) Google Scholar, 28Larance M. Kirkwood K.J. Xirodimas D.P. Lundberg E. Uhlen M. Lamond A.I. Characterization of MRFAP1 Turnover and interactions downstream of the NEDD8 pathway.Mol. Cell Proteomics. 2012; 11M111.014407Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Proteins were digested with trypsin and analyzed by LC-MS/MS. Three biological replicates of this experiment were performed. Peptides were identified and SILAC ratios quantitated using MaxQuant (24Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar, 25Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: A peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3450) Google Scholar). A total of over 74,000 peptides, corresponding to ∼5,000 proteins, were identified and quantified in at least two out of the three biological replicates (supplemental Table S1 and supplemental Table S2). The ∼5000 proteins analyzed in this study are highly representative of the human proteome, based on an analysis of amino acid frequencies and gene ontology (biological process) term frequencies in the total dataset (supplemental Fig. S1A, S1B). Analysis of the subcellular distribution of selected marker proteins known to be enriched in the isolated compartments (Fig. 1B), confirmed >80% enrichment of each fraction. For each subcellular fraction, proteins with masses from >500 kDa down to <10 kDa were efficiently size-separated using SDS-SEC (Fig. 1C). The workflow outlined above will detect rapidly depleted proteins (RDPs), whose abundance decreases following cycloheximide treatment, either because of degradation, or secretion. To identify RDPs with high statistical significance, p values were calculated using a t test of the replicate CHX/DMSO (M/L) SILAC ratios of each protein, compared with the CHX/DMSO (M/L) SILAC ratios for control, known stable proteins, i.e. β-Tubulin, Histone H2A, and GAPDH. A significance cutoff value of p < 0.05 (-Log10(p) > 1.3) was used, combined with a requirement of a >50% decrease in CHX/DMSO (M/L) SILAC ratio (Log2 normalized ratio CHX/DMSO < −1), indicating at least 50% depletion in less than 6 h. These criteria yielded 110 RDPs out of the total data set of ∼5000 proteins (Fig. 2a green box, and supplemental Table S3). This included proteins previously shown to be rapidly degraded, such as p21 (29Starostina N.G. Kipreos E.T. Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors.Trends Cell Biol. 2012; 22: 33-41Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the MORF4L1, and MRFAP1 proteins (28Larance M. Kirkwood K.J. Xirodimas D.P. Lundberg E. Uhlen M. Lamond A.I. Characterization of MRFAP1 Turnover and interactions downstream of the NEDD8 pathway.Mol. Cell Proteomics. 2012; 11M111.014407Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and Jun-B (30Gao M. Labuda T. Xia Y. Gallagher E. Fang D. Liu Y.C. Karin M. Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase itch.Science. 2004; 306: 271-275Crossref PubMed Scopus (336) Google Scholar) (Fig. 2A, green text). Some RDPs showed different degradation rates in separate subcellular compartments (supplemental Fig. S2), demonstrating that individual protein stability can vary depending on localization. The same significance cutoffs (p < 0.05 and >50% decrease in CHX/DMSO M/L SILAC ratio), were used to analyze the data from each subcellular fraction (Fig. 2B). This identified an additional subset of proteins whose abundance rapidly decreased in a specific compartment, but which were not identified as RDPs in the total data set (Fig. 2a,b). C7ORF59, FAM32A, PLOD3 and GLT25D1, provide clear examples of proteins not detected as statistically significant RDPs in the total data set, but where analysis of data from a single subcellular fraction show they behave as compartment-specific RDPs (Fig. 2A, 2B blue points and see supplemental Table S1 for more examples). These examples illustrate that this phenomenon is not confined to any one fraction alone and further show it applies to proteins detected in multiple locations and not only to proteins enriched in a single compartment. For most of the c
Referência(s)