Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Sep;79(3):1196–1205. doi: 10.1016/S0006-3495(00)76374-0

A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates.

H G Holzhütter 1, P M Kloetzel 1
PMCID: PMC1301016  PMID: 10968984

Abstract

There is now convincing evidence that the proteasome contributes to the generation of most of the peptides presented by major histocompatibility complex class I molecules. Here we present a model-based kinetic analysis of fragment patterns generated by the 20S proteasome from 20 to 40 residues long oligomeric substrates. The model consists of ordinary first-order differential equations describing the time evolution of the average probabilities with which fragments can be generated from a given initial substrate. First-order rate laws are used to describe the cleavage of peptide bonds and the release of peptides from the interior of the proteasome to the external space. Numerical estimates for the 27 unknown model parameters are determined across a set of five different proteins with known cleavage patterns. Testing the validity of the model by a jack knife procedure, about 80% of the observed fragments can be correctly identified, whereas the abundance of false-positive classifications is below 10%. From our theoretical approach, it is inferred that double-cleavage fragments of length 7-13 are predominantly cut out in "C-N-order" in that the C-terminus is generated first. This is due to striking differences in the further processing of the two fragments generated by the first cleavage. The upstream fragment exhibits a pronounced tendency to escape from second cleavage as indicated by a large release rate and a monotone exponential decline of peptide bond accessibility with increasing distance from the first scissile bond. In contrast, the release rate of the downstream fragment is about four orders of magnitude lower and the accessibility of peptide bonds shows a sharp peak in a distance of about nine residues from the first scissile bond. This finding strongly supports the idea that generation of fragments with well-defined lengths is favored in that temporary immobilization of the downstream fragment after the first cleavage renders it susceptible for a second cleavage.

Full Text

The Full Text of this article is available as a PDF (175.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baumeister W., Walz J., Zühl F., Seemüller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 1998 Feb 6;92(3):367–380. doi: 10.1016/s0092-8674(00)80929-0. [DOI] [PubMed] [Google Scholar]
  2. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. doi: 10.1016/0092-8674(94)90396-4. [DOI] [PubMed] [Google Scholar]
  3. Coux O., Tanaka K., Goldberg A. L. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 1996;65:801–847. doi: 10.1146/annurev.bi.65.070196.004101. [DOI] [PubMed] [Google Scholar]
  4. Dick L. R., Aldrich C., Jameson S. C., Moomaw C. R., Pramanik B. C., Doyle C. K., DeMartino G. N., Bevan M. J., Forman J. M., Slaughter C. A. Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides. J Immunol. 1994 Apr 15;152(8):3884–3894. [PMC free article] [PubMed] [Google Scholar]
  5. Eggers M., Boes-Fabian B., Ruppert T., Kloetzel P. M., Koszinowski U. H. The cleavage preference of the proteasome governs the yield of antigenic peptides. J Exp Med. 1995 Dec 1;182(6):1865–1870. doi: 10.1084/jem.182.6.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ehring B., Meyer T. H., Eckerskorn C., Lottspeich F., Tampé R. Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides. Eur J Biochem. 1996 Jan 15;235(1-2):404–415. doi: 10.1111/j.1432-1033.1996.00404.x. [DOI] [PubMed] [Google Scholar]
  7. Goldberg A. L., Gaczynska M., Grant E., Michalek M., Rock K. L. Functions of the proteasome in antigen presentation. Cold Spring Harb Symp Quant Biol. 1995;60:479–490. doi: 10.1101/sqb.1995.060.01.052. [DOI] [PubMed] [Google Scholar]
  8. Groettrup M., Soza A., Eggers M., Kuehn L., Dick T. P., Schild H., Rammensee H. G., Koszinowski U. H., Kloetzel P. M. A role for the proteasome regulator PA28alpha in antigen presentation. Nature. 1996 May 9;381(6578):166–168. doi: 10.1038/381166a0. [DOI] [PubMed] [Google Scholar]
  9. Groll M., Ditzel L., Löwe J., Stock D., Bochtler M., Bartunik H. D., Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997 Apr 3;386(6624):463–471. doi: 10.1038/386463a0. [DOI] [PubMed] [Google Scholar]
  10. Holzhütter H. G., Colosimo A. SIMFIT: a microcomputer software-toolkit for modelistic studies in biochemistry. Comput Appl Biosci. 1990 Jan;6(1):23–28. doi: 10.1093/bioinformatics/6.1.23. [DOI] [PubMed] [Google Scholar]
  11. Holzhütter H. G., Frömmel C., Kloetzel P. M. A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20 S proteasome. J Mol Biol. 1999 Mar 5;286(4):1251–1265. doi: 10.1006/jmbi.1998.2530. [DOI] [PubMed] [Google Scholar]
  12. Kisselev A. F., Akopian T. N., Goldberg A. L. Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes. J Biol Chem. 1998 Jan 23;273(4):1982–1989. doi: 10.1074/jbc.273.4.1982. [DOI] [PubMed] [Google Scholar]
  13. Kisselev A. F., Akopian T. N., Woo K. M., Goldberg A. L. 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. 1999 Feb 5;274(6):3363–3371. doi: 10.1074/jbc.274.6.3363. [DOI] [PubMed] [Google Scholar]
  14. Niedermann G., Butz S., Ihlenfeldt H. G., Grimm R., Lucchiari M., Hoschützky H., Jung G., Maier B., Eichmann K. Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity. 1995 Mar;2(3):289–299. doi: 10.1016/1074-7613(95)90053-5. [DOI] [PubMed] [Google Scholar]
  15. Niedermann G., King G., Butz S., Birsner U., Grimm R., Shabanowitz J., Hunt D. F., Eichmann K. The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8572–8577. doi: 10.1073/pnas.93.16.8572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nussbaum A. K., Dick T. P., Keilholz W., Schirle M., Stevanović S., Dietz K., Heinemeyer W., Groll M., Wolf D. H., Huber R. Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12504–12509. doi: 10.1073/pnas.95.21.12504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ossendorp F., Eggers M., Neisig A., Ruppert T., Groettrup M., Sijts A., Mengedë E., Kloetzel P. M., Neefjes J., Koszinowski U. A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation. Immunity. 1996 Aug;5(2):115–124. doi: 10.1016/s1074-7613(00)80488-4. [DOI] [PubMed] [Google Scholar]
  18. Pauletti G. M., Okumu F. W., Borchardt R. T. Effect of size and charge on the passive diffusion of peptides across Caco-2 cell monolayers via the paracellular pathway. Pharm Res. 1997 Feb;14(2):164–168. doi: 10.1023/a:1012040425146. [DOI] [PubMed] [Google Scholar]
  19. Shimbara N., Nakajima H., Tanahashi N., Ogawa K., Niwa S., Uenaka A., Nakayama E., Tanaka K. Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region. Genes Cells. 1997 Dec;2(12):785–800. doi: 10.1046/j.1365-2443.1997.1610359.x. [DOI] [PubMed] [Google Scholar]
  20. Sijts A. J., Ruppert T., Rehermann B., Schmidt M., Koszinowski U., Kloetzel P. M. Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes. J Exp Med. 2000 Feb 7;191(3):503–514. doi: 10.1084/jem.191.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stein R. L., Melandri F., Dick L. Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochemistry. 1996 Apr 2;35(13):3899–3908. doi: 10.1021/bi952262x. [DOI] [PubMed] [Google Scholar]
  22. Theobald M., Ruppert T., Kuckelkorn U., Hernandez J., Häussler A., Ferreira E. A., Liewer U., Biggs J., Levine A. J., Huber C. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J Exp Med. 1998 Sep 21;188(6):1017–1028. doi: 10.1084/jem.188.6.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang R., Chait B. T., Wolf I., Kohanski R. A., Cardozo C. Lysozyme degradation by the bovine multicatalytic proteinase complex (proteasome): evidence for a nonprocessive mode of degradation. Biochemistry. 1999 Nov 2;38(44):14573–14581. doi: 10.1021/bi990826h. [DOI] [PubMed] [Google Scholar]
  24. Wenzel T., Eckerskorn C., Lottspeich F., Baumeister W. Existence of a molecular ruler in proteasomes suggested by analysis of degradation products. FEBS Lett. 1994 Aug 1;349(2):205–209. doi: 10.1016/0014-5793(94)00665-2. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES