Skip to main content
SpringerLink
Log in

Characterization of extracellular proteasomes and its interacting proteins by iTRAQ mass spectrometry

  • Published:
Cell and Tissue Biology Aims and scope Submit manuscript

Abstract

We have performed the iTRAQ mass spectrometry analysis of extracellular proteasomes and revealed posttranslational modifications (PTMs) of subunits of extracellular 26S proteasomes. There are novel sites of ubiquitination and acetylation found in the proteasome subunits α2 (K196), α4 (K189 and K234), α6 (K217), and Rpn6 (A2). We have revealed that the extracellular proteasomes contain the standard 26S proteasome subunits as well as the PA200 regulator, previously believed to be located exclusively in the cell nuclei. We have additionally performed a primary screening of proteins interacting with extracellular proteasomes and found that associated with proteasomes are the proteins participating in such major cellular processes as transcription, DNA repair and translation, as well as cytoskeleton proteins and the ubiquitinproteasome system (UPS). Using the method of co-purification and immunoblotting we have confirmed interaction of proteasomes with nine proteins randomly chosen from the list of the identified extracellular proteasome partners.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

SDS:

sodium dodecyl sulfate

PAAG:

polyacrylamide gel

PTM:

posttranslational modifications

UPS:

ubiquitin-proteasome system

References

  • Albright, J.M., Romero, J., Saini, V., Sixt, S.U., Bird, M.D., Kovacs, E.J., Gamelli, R.L., Peters, J., and Majetschak, M., Proteasomes in human bronchoalveolar lavage fluid after burn and inhalation injury, J. Burn. Care Res., 2009, vol. 30, pp. 948–956.

    Article  PubMed  Google Scholar 

  • Besche, H.C., Haas, W., Gygi, S.P., and Goldberg, A.L., Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins, Biochemistry, 2009, vol. 48, pp. 2538–2549.

    Article  PubMed  CAS  Google Scholar 

  • Craig, R. and Beavis, R.C., TANDEM: matching proteins with tandem mass spectra, Bioinformatics, 2004, vol. 20, pp. 1466–1467.

    Article  PubMed  CAS  Google Scholar 

  • Fedorova, O.A., Moiseeva, T.N., Nikiforov, A.A., Tsimokha, A.S., Livinskaya, V.A., Hodson, M., Bottrill, A., Evteeva, I.N., Ermolayeva, J.B., Kuznetzova, I.M., Turoverov, K.K., Eperon, I., and Barlev, N.A., Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism, Biochem. Biophys. Res. Commun., 2011, vol. 416, pp. 258–265.

    Article  PubMed  CAS  Google Scholar 

  • Gazzah, A.C., Camoin, L., Abid, S., Bacha, H., and Ladjimi, M., iTRAQ: a method to elucidate cellular responses to mycotoxin zearalenone, J. Appl. Toxicol., 2012. doi: 10.1002/jat.1766

    Google Scholar 

  • Goldberg, A.L., Protein degradation and protection against misfolded or damaged proteins, Nature, 2003, vol. 426, pp. 895–899.

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Henry, L., Lavabre-Bertrand, T., Vercambre, L., Ramos, J., Carillo, S., Guiraud, I., Pouderoux, P., Bismuth, M., Valats, J.C., Demattei, C., Duny, Y., Chaze, I., Funakoshi, N., Bureau, J.P., Daurés, J.P., and Blanc, P., Plasma proteasome level is a reliable early marker of malignant transformation of liver cirrhosis, Gut, 2009, vol. 58, pp. 833–838.

    Article  PubMed  CAS  Google Scholar 

  • Hough, R., Pratt, G., and Rechsteiner, M., Purification of two high molecular weight proteases from rabbit reticulocyte lysates, J. Biol. Chem., 1987, vol. 262, pp. 8303–8313.

    PubMed  CAS  Google Scholar 

  • Kim, W., Bennett, E.J., Huttlin, E.L., Guo, A., Li, J., Possemato, A., Sowa, M.E., Rad, R., Rush, J., Comb, M.J., Harper, J.W., and Gygi, S.P., Systematic and quantitative assessment of the ubiquitin-modified proteome, Mol. Cell, 2011, vol. 44, pp. 325–340.

    Article  PubMed  CAS  Google Scholar 

  • Konstantinova, I.M., Tsimokha, A.S., and Mittenberg, A.G., Role of proteasomes in cellular regulation, Int. Rev. Cell Mol. Biol., 2008, vol. 267, pp. 59–124.

    Article  PubMed  Google Scholar 

  • Kulichkova, V.A., Mittenberg, A.G., Ermolaeva, Yu.B., Tsimokha, A.S., Volkova, I.V., Evteeva, I.N., Kozhukharova, I.V., Gauze, L.N., and Konstantinova, I.M., Specificity of the proteasome population secreted from cells into the culture medium, Dokl. Biol. Sci., 2004, vol. 399, pp. 503–506.

    Article  PubMed  CAS  Google Scholar 

  • Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685.

    Article  PubMed  CAS  Google Scholar 

  • Lavabre-Bertrand, T., Henry, L., Carillo, S., Guiraud, I., Ouali, A., Dutaud, D., Aubry, L., Rossi, J.F., and Bureau, J.P., Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies, Cancer, 2001, vol. 92, pp. 2493–2500.

    Article  PubMed  CAS  Google Scholar 

  • Meierhofer, D., Wang, X., Huang, L., and Kaiser, P., Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry, J. Proteome Res., 2008, vol. 7, pp. 4566–4576.

    Article  PubMed  CAS  Google Scholar 

  • Moiseeva, T.N., Mittenberg, A.G., and Barlev, N.A., Proteasomes and their role in transcriptional regulation, Tsitologiya, 2010a, vol. 52, no. 3, pp. 195–203.

    CAS  Google Scholar 

  • Moiseeva, T.N., Fedorova, O.A., Tsimokha, A.S., Mittenberg, A.G., and Barlev, N.A., Effect of ubiquitination on peptidase activities of proteasomes in genotoxic stress, Dokl. Biochem. Biophys., 2010b, vol. 435, pp. 307–312.

    Article  PubMed  CAS  Google Scholar 

  • Murakami, Y., Matsufuji, S., Hayashi, S., Tanahashi, N., and Tanaka, K., Degradation of ornithine decarboxylase by the 26S proteasome, Biochem. Biophys. Res. Commun., 2000, vol. 267, pp. 1–6.

    Article  PubMed  CAS  Google Scholar 

  • Perkins, D.N., Pappin, D.J.C., Creasy, D.M., and Cottrell, J.S., Probability-based protein identification by searching sequence databases using mass spectrometry data, Electrophoresis, 1999, vol. 20, pp. 3551–3567.

    Article  PubMed  CAS  Google Scholar 

  • Scanlon, T.C., Gottlieb, B., Durcan, T.M., Fon, E.A., Beitel, L.K., and Trifiro, M.A., Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method, Exp. Cell Res., 2009, vol. 315, pp. 176–189.

    Article  PubMed  CAS  Google Scholar 

  • Searle, B.C., Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies, Proteomics, 2010, vol. 10, pp. 1265–1269.

    Article  PubMed  CAS  Google Scholar 

  • Sixt, S.U. and Dahlmann, B., Extracellular, circulating proteasomes and ubiquitin-incidence and relevance, Biochim. Biophys. Acta, 2008, vol. 1782, pp. 817–823.

    Article  PubMed  CAS  Google Scholar 

  • Sixt, S.U. and Peters, J., Extracellular alveolar proteasome: possible role in lung injury and repair, Proc. Am. Thorac. Soc., 2010, vol. 7, pp. 91–96.

    Article  PubMed  Google Scholar 

  • Sixt, S.U., Beiderlinden, M., Jennissen, H.P., and Peters, J., Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?, Am. J. Physiol. Lung Cell Mol. Physiol., 2007, vol. 292, pp. 1280–1288.

    Article  Google Scholar 

  • Sixt, S.U., Adamzik, M., Spyrka, D., Saul, B., Hakenbeck, J., Wohlschlaeger, J., Costabel, U., Kloss, A., Giesebrecht, J., Dahlmann, B., and Peters, J., Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome, Am. J. Respir. Crit. Care Med., 2009, vol. 179, pp. 1098–1106.

    Article  PubMed  CAS  Google Scholar 

  • Stoebner, P.E., Lavabre-Bertrand, T., Henry, L., Guiraud, I., Carillo, S., Dandurand, M., Joujoux, J.M., Bureau, J.P., and Meunier, L., High plasma proteasome levels are detected in patients with metastatic malignant melanoma, Br. J. Dermatol., 2005, vol. 152, pp. 948–953.

    Article  PubMed  CAS  Google Scholar 

  • Tai, H.C., Besche, H., Goldberg, A.L., and Schuman, E.M., Characterization of the brain 26S proteasome and its interacting proteins, Front. Mol. Neurosci., 2010, vol. 3, pp. 12.

    PubMed  Google Scholar 

  • Touitou, R., Richardson, J., Bose, S., Nakanishi, M., Rivett, J., and Allday, M.J., 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., 2001, vol. 20, pp. 2367–2375.

    Article  PubMed  CAS  Google Scholar 

  • Tsimokha, A.S., Proteasomes: their role in cellular processes, Tsitologiya, 2010, vol. 52, no. 4, pp. 277–300.

    CAS  Google Scholar 

  • Wagner, S.A., Beli, P., Weinert, B.T., Nielsen, M.L., Cox, J., Mann, M., and Choudhary, C., A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles, Mol. Cell Proteomics, 2011, vol. 10, p. M111.013284.

    Article  PubMed  Google Scholar 

  • Zaikova, Yu.Ya., Kulichkova, V.A., Ermolaeva, Yu.B., Gauze, L.N., and Tsimokha, A.S., Comparative analysis of extra- and intracellular proteasomes from K562 cells, Cell Tiss. Biol., 2011, vol. 5, no. 5, pp. 480–486.

    Article  Google Scholar 

  • Zhang, M., Pickart, C.M., and Coffino, P., Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate, EMBO J., 2003, vol. 22, pp. 1488–1496.

    Article  PubMed  CAS  Google Scholar 

  • Zoeger, A., Blau, M., Egerer, K., Feist, E., and Dahlmann, B., Circulating proteasomes are functional and have a subtype pattern distinct from 20S proteasomes in major blood cells, Clin. Chem., 2006, vol. 52, pp. 2079–2086.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

    Yu. Ya. Zaikova, V. A. Kulichkova, Yu. B. Ermolaeva, N. A. Barlev & A. S. Tsimokha

  2. St. Petersburg State Polytechnical University, St. Petersburg, Russia

    Yu. Ya. Zaikova

  3. University of Leicester, Leicester, UK

    A. Bottrill & N. A. Barlev

  4. St. Petersburg State Institute of Technology, St. Petersburg, Russia

    N. A. Barlev

Authors
  1. Yu. Ya. Zaikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. A. Kulichkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu. B. Ermolaeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Bottrill
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. A. Barlev
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. S. Tsimokha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. S. Tsimokha.

Additional information

Original Russian Text © Yu.Ya. Zaikova, V.A. Kulichkova, Yu.B. Ermolaeva, A. Bottrill, N.A. Barlev, A.S. Tsimokha, 2013, published in Tsitologiya, Vol. 55, No. 2, 2013, pp. 111–122.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zaikova, Y.Y., Kulichkova, V.A., Ermolaeva, Y.B. et al. Characterization of extracellular proteasomes and its interacting proteins by iTRAQ mass spectrometry. Cell Tiss. Biol. 7, 253–265 (2013). https://doi.org/10.1134/S1990519X13030139

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1990519X13030139

Keywords

Search

Navigation