SpringerProtocols: Abstract: Assays of Proteasome-Dependent Cleavage Products

Assays of Proteasome-Dependent Cleavage Products

By: Stefan Tenzer³, Hansjörg Schild⁴

Abstract

Full Text

Download PDF (467K)

The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3–20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The obtained fractions are further analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation, which allows a quantitative analysis of the digestion products.

Affiliation(s): (3) Institute of Immunology, University of Mainz, Mainz, Germany
(4) Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany

Book Title: Ubiquitin-Proteasome Protocols

Series: Methods in Molecular Biology | Volume: 301 | Pub. Date: Apr-01-2005 | Page Range: 97-115 | DOI: 10.1385/1-59259-895-1:097

Subject: Biochemistry

Key Words: Analysis - antigen processing - Edman degradation - in vitro degradation - MALDI-mass spectrometry - peptide - proteasome - purification - quantification

References

Download References

1.	Kisselev, A. F., Akopian, T. N., Woo, K. M., and Goldberg, A. L. (1999) The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J. Biol. Chem. 274, 3363–3371.

2.	Rock, K. L. and Goldberg, A. L. (1999) Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17, 739–779.

3.	Groll, M., Ditzel, L., Lowe, J., et al. (1997) Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463–471.

4.	Lowe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W., and Huber, R. (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539.

5.	Groettrup, M., Ruppert, T., Kuehn, L., et al. (1995) The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro. J. Biol. Chem. 270, 23808–23815.

6.	Eleuteri, A. M., Kohanski, R. A., Cardozo, C., and Orlowski, M. (1997) Bovine spleen multicatalytic proteinase complex (proteasome): replacement of X, Y, and Z subunits by LMP7, LMP2, and MECL1 and changes in properties and specificity. J. Biol. Chem. 272, 11824–11831.

7.	Boes, B., Hengel, H., Ruppert, T., Multhaup, G., Koszinowski, U. H., and Kloetzel, P. M. (1994) Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. J. Exp. Med. 179, 901–909.

8.	Gaczynska, M., Rock, K. L., and Goldberg, A. L. (1993) Gamma-interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365, 264–267.

9.	Cardozo, C. and Kohanski, R. A. (1998) Altered properties of the branched chain amino acid-preferring activity contribute to increased cleavages after branched chain residues by the “immunoproteasome.” J. Biol. Chem. 273, 16764–16770.

10.	Sijts, A. J., Standera, S., Toes, R. E., et al. (2000) MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells. J. Immunol. 164, 4500–4506.

11.	Sijts, A. J., Ruppert, T., Rehermann, B., Schmidt, M., Koszinowski, U., and Kloetzel, P. M. (2000) Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes. J. Exp. Med. 191, 503–514.

12.	van Hall, T., Sijts, A., Camps, M., et al. (2000) Differential influence on cytotoxic T lymphocyte epitope presentation by controlled expression of either proteasome immunosubunits or PA28. J. Exp. Med. 192, 483–494.

13.	Schwarz, K., van den, B. M., Kostka, S., et al. (2000) Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope. J. Immunol. 165, 768–778.

14.	Morel, S., Levy, F., Burlet-Schiltz, O., et al. (2000) Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity 12, 107–117. <Occurrence Type="DOI"><Handle>10.1016/S1074-7613(00)80163-6</Handle></Occurrence>

15.	Nussbaum, A. K., Kuttler, C., Tenzer, S., and Schild, H. (2003) Using the World Wide Web for predicting CTL epitopes. Curr. Opin. Immunol. 15, 69–74.

16.	Ayyoub, M., Stevanovic, S., Sahin, U., et al. (2002) Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma. J. Immunol. 168, 1717–1722.

17.	Kessler, J. H., Beekman, N. J., Bres-Vloemans, S. A., et al. (2001) Efficient identification of novel HLA-A()0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis. J. Exp. Med.* 193, 73–88.

18.	Emmerich, N. P., Nussbaum, A. K., Stevanovic, S., et al. (2000) The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate. J. Biol. Chem. 275, 21140–21148.

19.	Toes, R. E., Nussbaum, A. K., Degermann, S., et al. (2001) Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products. J. Exp. Med. 194, 1–12.

20.	Kuttler, C., Nussbaum, A. K., Dick, T. P., Rammensee, H. G., Schild, H., and Hadeler, K. P. (2000) An algorithm for the prediction of proteasomal cleavages. J. Mol. Biol. 298, 417–429.

21.	Nussbaum, A. K., Kuttler, C., Hadeler, K. P., Rammensee, H. G., and Schild, H. (2001) PAProC: a prediction algorithm for proteasomal cleavages available on the WWW. Immunogenetics 53, 87–94.

22.	Kesmir, C., Nussbaum, A. K., Schild, H., Detours, V., and Brunak, S. (2002) Prediction of proteasome cleavage motifs by neural networks. Prot. Eng. 15, 287–296.

23.	Holzhutter, H. G., Frommel, C., and Kloetzel, P. M. (1999) A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20 S proteasome. J. Mol. Biol. 286, 1251–1265.

24.	Stevanovic, S. and Jung, G. (1993) Multiple sequence analysis: pool sequencing of synthetic and natural peptide libraries. Anal. Biochem. 212, 212–220.

Comments (Loading...)

Post comment/View all comments