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Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells

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Abstract

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin–proteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC–ESI–MS. Taking the results of all experiments together, no quantitative changes were observed for the α- and β-subunits of the 20S proteasome except for subunit α7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (α7a) and up-regulation of the more basic protein species (α7b) during apoptosis. The difference in these two α7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in α7a, whereas these modifications were missing in α7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit α7 after induction of apoptosis.

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Abbreviations

19S-Reg:

19S regulator complex

5-FU:

5-Fluorouracil

H/L:

Heavy to light

PARP1:

Poly (ADP-ribose) polymerase-1

SILAC:

Stable isotope labeling with amino acids in cell culture

References

  • Adrain C, Creagh EM, Cullen SP, Martin SJ (2004) Caspase-dependent inactivation of proteasome function during programmed cell death in Drosophila and man. J Biol Chem 279:36923–36930

    Article  PubMed  CAS  Google Scholar 

  • Alvarez-Castelao B, Castano JG (2005) Mechanism of direct degradation of IkappaBalpha by 20S proteasome. FEBS Lett 579:4797–4802

    Article  PubMed  CAS  Google Scholar 

  • Arrigo AP, Mehlen P (1993) Hela cells proteasome interacts with leucine-rich polypeptides and contains a phosphorylated subunit. Biochem Biophys Res Commun 194:1387–1393

    Article  PubMed  CAS  Google Scholar 

  • Bardag-Gorce F, Venkatesh R, Li J, French BA, French SW (2004) Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared. Life Sci 75:585–597

    Article  PubMed  CAS  Google Scholar 

  • Baumeister W, Walz J, Zuhl F, Seemuller E (1998) The proteasome: paradigm of a self-compartmentalizing protease. Cell 92:367–380

    Article  PubMed  CAS  Google Scholar 

  • Bochtler M, Ditzel L, Groll M, Hartmann C, Huber R (1999) The proteasome. Annu Rev Biophys Biomol Struct 28:295–317

    Article  PubMed  CAS  Google Scholar 

  • Boelens WC, Croes Y, de Jong WW (2001) Interaction between alphaB-crystallin and the human 20S proteasomal subunit C8/alpha7. Biochim Biophys Acta 1544:311–319

    Article  PubMed  CAS  Google Scholar 

  • Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ (2004) Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon. Biochem J 378:177–184

    Article  PubMed  CAS  Google Scholar 

  • Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333

    Article  PubMed  CAS  Google Scholar 

  • Castano JG, Mahillo E, Arizti P, Arribas J (1996) Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis. Biochemistry 35:3782–3789

    Article  PubMed  CAS  Google Scholar 

  • Claverol S, Burlet-Schiltz O, Girbal-Neuhauser E, Gairin JE, Monsarrat B (2002) Mapping and structural dissection of human 20 S proteasome using proteomic approaches. Mol Cell Proteomics 1:567–578

    Article  PubMed  CAS  Google Scholar 

  • Creasy DM, Cottrell JS (2002) Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2:1426–1434

    Article  PubMed  CAS  Google Scholar 

  • Dahlmann B (2005) Proteasomes. Essays Biochem 41:31–48

    Article  PubMed  CAS  Google Scholar 

  • Dahlmann B, Ruppert T, Kuehn L, Merforth S, Kloetzel PM (2000) Different proteasome subtypes in a single tissue exhibit different enzymatic properties. J Mol Biol 303:643–653

    Article  PubMed  CAS  Google Scholar 

  • Doherty NS, Littman BH, Reilly K, Swindell AC, Buss JM, Anderson NL (1998) Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis. Electrophoresis 19:355–363

    Article  PubMed  CAS  Google Scholar 

  • Dong J, Chen W, Welford A, Wandinger-Ness A (2004) The proteasome alpha-subunit XAPC7 interacts specifically with Rab7 and late endosomes. J Biol Chem 279:21334–21342

    Article  PubMed  CAS  Google Scholar 

  • Drews O, Wildgruber R, Zong C, Sukop U, Nissum M, Weber G, Gomes AV, Ping P (2007) Mammalian proteasome subpopulations with distinct molecular compositions and proteolytic activities. Mol Cell Proteomics 6:2021–2031

    Article  PubMed  CAS  Google Scholar 

  • Froment C, Uttenweiler-Joseph S, Bousquet-Dubouch MP, Matondo M, Borges JP, Esmenjaud C, Lacroix C, Monsarrat B, Burlet-Schiltz O (2005) A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity. Proteomics 5:2351–2363

    Article  PubMed  CAS  Google Scholar 

  • Fujiwara N, Nakano M, Kato S, Yoshihara D, Ookawara T, Eguchi H, Taniguchi N, Suzuki K (2007) Oxidative modification to cysteine sulfonic acid of Cys111 in human copper–zinc superoxide dismutase. J Biol Chem 282:35933–35944

    Article  PubMed  CAS  Google Scholar 

  • Glickman MH, Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428

    PubMed  CAS  Google Scholar 

  • Grubbs FE (1969) Procedures for detecting outlying observations in samples. Technometrics 11:1–21

    Article  Google Scholar 

  • Guerrero C, Tagwerker C, Kaiser P, Huang L (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol Cell Proteomics 5:366–378

    PubMed  CAS  Google Scholar 

  • Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479

    Article  PubMed  CAS  Google Scholar 

  • Huang L, Burlingame AL (2005) Comprehensive mass spectrometric analysis of the 20S proteasome complex. Methods Enzymol 405:187–236

    Article  PubMed  CAS  Google Scholar 

  • Ikeda K, Nakano R, Uraoka M, Nakagawa Y, Koide M, Katsume A, Minamino K, Yamada E, Yamada H, Quertermous T, Matsubara H (2009) Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2. Proc Natl Acad Sci USA 106:8227–8232

    Article  PubMed  CAS  Google Scholar 

  • Iwafune Y, Kawasaki H, Hirano H (2004) Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry. Arch Biochem Biophys 431:9–15

    Article  PubMed  CAS  Google Scholar 

  • Jin Y, Lee H, Zeng SX, Dai MS, Lu H (2003) MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation. EMBO J 22:6365–6377

    Article  PubMed  CAS  Google Scholar 

  • Jungblut PR, Seifert R (1990) Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. J Biochem Biophys Methods 21:47–58

    Article  PubMed  CAS  Google Scholar 

  • Jungblut PR, Holzhutter HG, Apweiler R, Schluter H (2008) The speciation of the proteome. Chem Cent J 2:16

    Article  PubMed  Google Scholar 

  • Keller DM, Zeng X, Wang Y, Zhang QH, Kapoor M, Shu H, Goodman R, Lozano G, Zhao Y, Lu H (2001) A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol Cell 7:283–292

    Article  PubMed  CAS  Google Scholar 

  • Kimura Y, Takaoka M, Tanaka S, Sassa H, Tanaka K, Polevoda B, Sherman F, Hirano H (2000) N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome. J Biol Chem 275:4635–4639

    Article  PubMed  CAS  Google Scholar 

  • Koehler CJ, Strozynski M, Kozielski F, Treumann A, Thiede B (2009) Isobaric peptide termini labeling for MS/MS-based quantitative proteomics. J Proteome Res 8:4333–4341

    Article  PubMed  CAS  Google Scholar 

  • Kuckelkorn U, Ruppert T, Strehl B, Jungblut PR, Zimny-Arndt U, Lamer S, Prinz I, Drung I, Kloetzel PM, Kaufmann SH, Steinhoff U (2002) Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity. J Exp Med 195:983–990

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lehmann WD, Kruger R, Salek M, Hung CW, Wolschin F, Weckwerth W (2007) Neutral loss-based phosphopeptide recognition: a collection of caveats. J Proteome Res 6:2866–2873

    Article  PubMed  CAS  Google Scholar 

  • Lim JC, Choi HI, Park YS, Nam HW, Woo HA, Kwon KS, Kim YS, Rhee SG, Kim K, Chae HZ (2008) Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity. J Biol Chem 283:28873–28880

    Article  PubMed  CAS  Google Scholar 

  • Ling MT, Chiu YT, Lee TK, Leung SC, Fung MK, Wang X, Wong KF, Wong YC (2008) Id-1 induces proteasome-dependent degradation of the HBX protein. J Mol Biol 382:34–43

    Article  PubMed  CAS  Google Scholar 

  • Liu CH, Goldberg AL, Qiu XB (2007) New insights into the role of the ubiquitin–proteasome pathway in the regulation of apoptosis. Chang Gung Med J 30:469–479

    PubMed  Google Scholar 

  • Lu H, Zong C, Wang Y, Young GW, Deng N, Souda P, Li X, Whitelegge J, Drews O, Yang PY, Ping P (2008) Revealing the dynamics of the 20 S proteasome phosphoproteome: a combined CID and electron transfer dissociation approach. Mol Cell Proteomics 7:2073–2089

    Article  PubMed  CAS  Google Scholar 

  • Ludemann R, Lerea KM, Etlinger JD (1993) Copurification of casein kinase II with 20 S proteasomes and phosphorylation of a 30-kDa proteasome subunit. J Biol Chem 268:17413–17417

    PubMed  CAS  Google Scholar 

  • Mason GG, Hendil KB, Rivett AJ (1996) Phosphorylation of proteasomes in mammalian cells. Identification of two phosphorylated subunits and the effect of phosphorylation on activity. Eur J Biochem 238:453–462

    Article  PubMed  CAS  Google Scholar 

  • Mittenberg AG, Moiseeva TN, Barlev NA (2008) Role of proteasomes in transcription and their regulation by covalent modifications. Front Biosci 13:7184–7192

    Article  PubMed  CAS  Google Scholar 

  • Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021

    Article  PubMed  CAS  Google Scholar 

  • Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  PubMed  CAS  Google Scholar 

  • Orlowski RZ, Kuhn DJ (2008) Proteasome inhibitors in cancer therapy: lessons from the first decade. Clin Cancer Res 14:1649–1657

    Article  PubMed  CAS  Google Scholar 

  • Orlowski M, Wilk S (2003) Ubiquitin-independent proteolytic functions of the proteasome. Arch Biochem Biophys 415:1–5

    Article  PubMed  CAS  Google Scholar 

  • Paradela A, Albar JP (2008) Advances in the analysis of protein phosphorylation. J Proteome Res 7:1809–1818

    Article  PubMed  CAS  Google Scholar 

  • Pardo PS, Murray PF, Walz K, Franco L, Passeron S (1998) In vivo and in vitro phosphorylation of the alpha 7/PRS1 subunit of Saccharomyces cerevisiae 20 S proteasome: in vitro phosphorylation by protein kinase CK2 is absolutely dependent on polylysine. Arch Biochem Biophys 349:397–401

    Article  PubMed  CAS  Google Scholar 

  • Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

    Article  PubMed  CAS  Google Scholar 

  • Rabl J, Smith DM, Yu Y, Chang SC, Goldberg AL, Cheng Y (2008) Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol Cell 30:360–368

    Article  PubMed  CAS  Google Scholar 

  • Raijmakers R, Berkers CR, de Jong A, Ovaa H, Heck AJ, Mohammed S (2008) Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity. Mol Cell Proteomics 7:1755–1762

    Article  PubMed  CAS  Google Scholar 

  • Rechsteiner M, Hill CP (2005) Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol 15:27–33

    Article  PubMed  CAS  Google Scholar 

  • Rivett AJ, Mason GG, Thomson S, Pike AM, Savory PJ, Murray RZ (1995) Catalytic components of proteasomes and the regulation of proteinase activity. Mol Biol Rep 21:35–41

    Article  PubMed  CAS  Google Scholar 

  • Schluter H, Apweiler R, Holzhutter HG, Jungblut PR (2009) Finding one’s way in proteomics: a protein species nomenclature. Chem Cent J 3:11

    Article  PubMed  Google Scholar 

  • Schmidt F, Dahlmann B, Janek K, Kloss A, Wacker M, Ackermann R, Thiede B, Jungblut PR (2006) Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches. Proteomics 6:4622–4632

    Article  PubMed  CAS  Google Scholar 

  • Schmidt F, Strozynski M, Salus SS, Nilsen H, Thiede B (2007) Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC). Rapid Commun Mass Spectrom 21:3919–3926

    Article  PubMed  CAS  Google Scholar 

  • Schmidt F, Fiege T, Hustoft HK, Kneist S, Thiede B (2009) Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI-MS. Proteomics 9:1994–2003

    Article  PubMed  CAS  Google Scholar 

  • Sdek P, Ying H, Chang DL, Qiu W, Zheng H, Touitou R, Allday MJ, Xiao ZX (2005) MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein. Mol Cell 20:699–708

    Article  PubMed  CAS  Google Scholar 

  • Shu F, Guo S, Dang Y, Qi M, Zhou G, Guo Z, Zhang Y, Wu C, Zhao S, Yu L (2003) Human aurora-B binds to a proteasome alpha-subunit HC8 and undergoes degradation in a proteasome-dependent manner. Mol Cell Biochem 254:157–162

    Article  PubMed  CAS  Google Scholar 

  • Sun XM, Butterworth M, MacFarlane M, Dubiel W, Ciechanover A, Cohen GM (2004) Caspase activation inhibits proteasome function during apoptosis. Mol Cell 14:81–93

    Article  PubMed  CAS  Google Scholar 

  • Tanaka K, Tanahashi N, Tsurumi C, Yokota KY, Shimbara N (1997) Proteasomes and antigen processing. Adv Immunol 64:1–38

    Article  PubMed  CAS  Google Scholar 

  • Thiede B, Hohenwarter W, Krah A, Mattow J, Schmid M, Schmidt F, Jungblut PR (2005) Peptide mass fingerprinting. Methods 35:237–247

    Article  PubMed  CAS  Google Scholar 

  • Touitou R, Richardson J, Bose S, Nakanishi M, Rivett J, Allday MJ (2001) A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome. EMBO J 20:2367–2375

    Article  PubMed  CAS  Google Scholar 

  • Tsimokha AS, Mittenberg AG, Kulichkova VA, Kozhukharova IV, Gause LN, Konstantinova IM (2007) Changes in composition and activities of 26S proteasomes under the action of doxorubicin-apoptosis inductor of erythroleukemic K562 cells. Cell Biol Int 31:338–348

    Article  PubMed  CAS  Google Scholar 

  • Uttenweiler-Joseph S, Claverol S, Sylvius L, Bousquet-Dubouch MP, Burlet-Schiltz O, Monsarrat B (2008) Toward a full characterization of the human 20S proteasome subunits and their isoforms by a combination of proteomic approaches. Methods Mol Biol 484:111–130

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Chen CF, Baker PR, Chen PL, Kaiser P, Huang L (2007) Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46:3553–3565

    Article  PubMed  CAS  Google Scholar 

  • Wehren A, Meyer HE, Sobek A, Kloetzel PM, Dahlmann B (1996) Phosphoamino acids in proteasome subunits. Biol Chem 377:497–503

    PubMed  CAS  Google Scholar 

  • Yi P, Feng Q, Amazit L, Lonard DM, Tsai SY, Tsai MJ, O’Malley BW (2008) Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1. Mol Cell 29:465–476

    Article  PubMed  CAS  Google Scholar 

  • Ying J, Clavreul N, Sethuraman M, Adachi T, Cohen RA (2007) Thiol oxidation in signaling and response to stress: detection and quantification of physiological and pathophysiological thiol modifications. Free Radic Biol Med 43:1099–1108

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Torii N, Furusaka A, Malayaman N, Hu Z, Liang TJ (2000) Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex. J Biol Chem 275:15157–15165

    Article  PubMed  CAS  Google Scholar 

  • Zimny-Arndt U, Schmid M, Ackermann R, Jungblut PR (2009) Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry. Methods Mol Biol 492:65–91

    Article  PubMed  CAS  Google Scholar 

  • Zong C, Young GW, Wang Y, Lu H, Deng N, Drews O, Ping P (2008) Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes. Proteomics 8:5025–5037

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The present study was supported by the National Program for Research in Functional Genomics in Norway (FUGE, project no. 183418/S10) of the Norwegian Research Council to BT.

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Authors and Affiliations

  1. The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, Blindern, P.O. Box 1125, 0317, Oslo, Norway

    Frank Schmidt, Hanne K. Hustoft, Christian J. Koehler, Margarita Strozynski & Bernd Thiede

  2. Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

    Frank Schmidt

  3. Institute of Biochemistry, Charité-Universitätsmedizin, Berlin, Germany

    Burkhardt Dahlmann & Alexander Kloß

  4. Max Planck Institute for Infection Biology, Core Facility Protein Analysis, Berlin, Germany

    Ursula Zimny-Arndt & Peter R. Jungblut

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  1. Frank Schmidt
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  2. Burkhardt Dahlmann
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Correspondence to Bernd Thiede.

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This article is published as part of the Special Issue on Protein Species and Time Schedule.

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Schmidt, F., Dahlmann, B., Hustoft, H.K. et al. Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells. Amino Acids 41, 351–361 (2011). https://doi.org/10.1007/s00726-010-0575-6

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