Prediction Algorithm for Proteasomal Cleavages

PAProC's location within the MHC class I antigen processing pathway.

Background information

Page content:  
Importance for immune response    
Purpose of a prediction tool    

What are proteasomes ?
Proteasomes are cytosolic multisubunit proteases which are involved in cell cycle control, transcription factor activation and the generation of peptide ligands for MHC I molecules (for reviews, see Baumeister et al. (1998), Rock & Goldberg (1999), Uebel & Tampe (1999)). They exist in several forms; either as proteolytically active core complexes or 20S proteasomes and, when associated with the ATP-dependent 19S cap complexes, larger 26S proteasomes that are able to recognize proteins marked by ubiquitin for proteasomal degradation (Jentsch & Schlenker, 1995; Hershko & Ciechanover, 1998). Another protein complex known to associate with the 20S core particle is PA28, the 11S regulator (Ahn et al., 1995), which was shown to improve the yield of antigenic peptides (Groettrup et al., 1996; Dick et al., 1996).
Eukaryotic 20S proteasomes consist of four stacked rings (overall stoichiometry alpha7beta7beta7alpha7), each consisting of 7 different subunits (Groll et al., 1997 [See picture taken from this reference at the bottom of the page. The picture shows a section through the cylinder of yeast 20S proteasomes. The positions of the active sites are highlighted through binding of specific inhibitors (yellow).]) . Each of the two inner beta-rings carries three catalytically active sites on its inner surface. Their proteolytic specificities have been described as chymotrypsin-like (cleaving after large, hydrophobic AAs), trypsin-like (cleaving after basic AAs) and peptidyl-glutamyl-peptide-hydrolyzing (cleaving after acidic AAs) (for review, see Uebel & Tampe (1999)). Strings of unfolded proteins are thought to be inserted into the cylinder and to be cut into pieces by the active sites; the resulting peptide fragments are then released into the cytosol. Functionally, proteasomal protein degradation is believed to proceed from one substrate end to the other ("processively"), without the release of large degradation intermediates (Akopian et al., 1997; Nussbaum et al., 1998; Kisselev et al., 1999).

Why is proteasomal cleavage specificity important for immune responses?
In vertebrate cells, some of the proteolytic fragments produced by proteasomes are fed into the antigen processing machinery (see picture ). Since peptide presentation by MHC I molecules at the cell surface is an intrinsic requirement for the ability of the immune system to eradicate virus-infected or transformed cells (Rammensee et al., 1993; Pamer & Cresswell, 1998), it is of general interest to know exactly how the proteasome is involved in this process. Proteasomal cleavage specificity has been assessed by in vitro digestion experiments using either tri- or tetrapeptides with fluorogenic leaving groups (Kuckelkorn et al., 1995; Heinemeyer et al., 1997; Arendt & Hochstrasser, 1997), peptides of 15-40 AAs (Boes et al., 1994; Niedermann et al., 1995; Niedermann et al., 1996; Dick et al., 1998), or denatured proteins (Dick et al., 1991; Dick et al., 1994; Kisselev et al., 1998, Kisselev et al., 1999) as substrates. We analyzed the cleavage preferences of yeast wild-type and mutant proteasomes in a non-modified protein (Nussbaum et al., 1998). Using statistical analysis of cut sites, it was possible for the first time to determine so-called cleavage motifs, i.e. the preferred sequences around cleavage sites, for the three active beta-subunits of yeast proteasomes.

Why would a prediction tool be beneficial?
In order to apply experimentally determined information on cleavage site selection by proteasomes to any possible proteasome substrate, one needs an automated prediction device. Such devices already exist for the binding of peptides to MHC I molecules (Database SYFPEITHI , Rammensee et al., 1997) and have been described for peptide transport by the transporter associated with antigen processing (TAP) (Daniel et al., 1998). However, devices for the prediction of proteasomal cleavages are only at the beginning of their development. A proteasomal cleavage prediction tool could, especially in combination with MHC ligand predictors as SYFPEITHI, help to improve the forecast of MHC class I restricted CTL-responses. More specifically, it could support researchers in their quest for individual CTL-epitopes by limiting the number of possible MHC class I ligands from protein antigens. In addition, the effect of amino acid mutations in viral or tumor-specific proteins on antigen presentation could be assessed. Thus, proteasomal cleavage prediction would lend a hand in rational vaccine design.

We have made the first step towards this end by providing PAProC (Prediction Algorithm for Proteasomal Cleavages), a public prediction tool for proteasomal cleavages. PAProC offers information on both the general cleavability of amino acid sequences (cuts per amino acids) and individual cleavages (positions and estimated strength; for details, please refer to the user information).
PAProC was developed from the beginning, i.e. from the experimental basis to the ready-to-use public prediction tool, by proteasome experts at the Department of Immunology in close collaboration with programmers at the Department of Biomathematics, both at the University of Tübingen, Germany. We are therefore confident that PAProC has profited from the best possible expertise. However, we are aware of the fact that PAProC is still in its teething stage. For example, cleavage sites and estimated cleavage strength are not yet based on quantified cleavage data (in PAProC I). Therefore, we are continuously working to improve PAProC. However, we need your help: The program will profit from your experience with it. So please let us know how PAProC performed for you. Thank you for your collaboration.

On our link list you can find several pages concerning proteasome and more.

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Last update: 13.4.2005

More detailed information about this program can be found in the following publications:
C. Kuttler, A.K. Nussbaum, T.P. Dick, H.-G. Rammensee, H. Schild, K.P. Hadeler, An algorithm for the prediction of proteasomal cleavages, J. Mol. Biol. 298 (2000), 417-429 , and
A.K. Nussbaum, C. Kuttler, K.P. Hadeler, H.-G. Rammensee, H. Schild, PAProC: A Prediction Algorithm for Proteasomal Cleavages available on the WWW, Immunogenetics 53 (2001), 87-94
For comprehensive background information, please refer to From the test tube to the World Wide Web - The cleavage specificity of the proteasome (A.K. Nussbaum, dissertation, University of Tuebingen, Germany, 2001).
The use of PAProC is restricted to non-commercial purposes.
Inner surface of the yeast 20S proteasome; yellow:
inhibitors bound to the three active sites [Groll et al. (1997), Nature].