Logo of biophysjLink to Publisher's site
Biophys J. 2000 Sep; 79(3): 1196–1205.
PMCID: PMC1301016

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


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 (175K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baumeister W, Walz J, Zühl F, Seemüller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 1998 Feb 6;92(3):367–380. [PubMed]
  • Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. [PubMed]
  • Coux O, Tanaka K, Goldberg AL. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 1996;65:801–847. [PubMed]
  • Dick LR, Aldrich C, Jameson SC, Moomaw CR, Pramanik BC, Doyle CK, DeMartino GN, Bevan MJ, Forman JM, Slaughter CA. 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]
  • Eggers M, Boes-Fabian B, Ruppert T, Kloetzel PM, Koszinowski UH. The cleavage preference of the proteasome governs the yield of antigenic peptides. J Exp Med. 1995 Dec 1;182(6):1865–1870. [PMC free article] [PubMed]
  • Ehring B, Meyer TH, 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. [PubMed]
  • Goldberg AL, Gaczynska M, Grant E, Michalek M, Rock KL. Functions of the proteasome in antigen presentation. Cold Spring Harb Symp Quant Biol. 1995;60:479–490. [PubMed]
  • Groettrup M, Soza A, Eggers M, Kuehn L, Dick TP, Schild H, Rammensee HG, Koszinowski UH, Kloetzel PM. A role for the proteasome regulator PA28alpha in antigen presentation. Nature. 1996 May 9;381(6578):166–168. [PubMed]
  • Groll M, Ditzel L, Löwe J, Stock D, Bochtler M, Bartunik HD, Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997 Apr 3;386(6624):463–471. [PubMed]
  • Holzhütter HG, Colosimo A. SIMFIT: a microcomputer software-toolkit for modelistic studies in biochemistry. Comput Appl Biosci. 1990 Jan;6(1):23–28. [PubMed]
  • Holzhütter HG, Frömmel C, Kloetzel PM. 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. [PubMed]
  • Kisselev AF, Akopian TN, Goldberg AL. 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. [PubMed]
  • Kisselev AF, Akopian TN, Woo KM, Goldberg AL. 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. [PubMed]
  • Niedermann G, Butz S, Ihlenfeldt HG, 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. [PubMed]
  • Niedermann G, King G, Butz S, Birsner U, Grimm R, Shabanowitz J, Hunt DF, 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. [PMC free article] [PubMed]
  • Nussbaum AK, Dick TP, Keilholz W, Schirle M, Stevanović S, Dietz K, Heinemeyer W, Groll M, Wolf DH, Huber R, et al. 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. [PMC free article] [PubMed]
  • Ossendorp F, Eggers M, Neisig A, Ruppert T, Groettrup M, Sijts A, Mengedë E, Kloetzel PM, Neefjes J, Koszinowski U, et al. 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. [PubMed]
  • Pauletti GM, Okumu FW, Borchardt RT. 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. [PubMed]
  • 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. [PubMed]
  • Sijts AJ, Ruppert T, Rehermann B, Schmidt M, Koszinowski U, Kloetzel PM. 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. [PMC free article] [PubMed]
  • Stein RL, Melandri F, Dick L. Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochemistry. 1996 Apr 2;35(13):3899–3908. [PubMed]
  • Theobald M, Ruppert T, Kuckelkorn U, Hernandez J, Häussler A, Ferreira EA, Liewer U, Biggs J, Levine AJ, Huber C, et al. 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. [PMC free article] [PubMed]
  • Wang R, Chait BT, Wolf I, Kohanski RA, 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. [PubMed]
  • 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. [PubMed]

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


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...