Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously.We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

We present a predictive method that can simulate an essential step in the antigen presentation in higher vertebrates, namely the step involving the proteasomal degradation of polypeptides into fragments which have the potential to bind to MHC Class I molecules. Proteasomal cleavage prediction algorithms published so far were trained on data from in vitro digestion experiments with constitutive proteasomes. As a result, they did not take into account the characteristics of the structurally modified proteasomes--often called immunoproteasomes--found in cells stimulated by gamma-interferon under physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC Class I ligands, i.e. the specificity of the immunoproteasome is different than the constitutive proteasome. The tools developed in this study in combination with a predictor of MHC and TAP binding capacity should give a more complete prediction of the generation and presentation of peptides on MHC Class I molecules. Here we demonstrate that such an approach produces an accurate prediction of the CTL the epitopes in HIV Nef. The method is available at www.cbs.dtu.dk/services/NetChop/.

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active ␤ subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.The eukaryotic 20S proteasome represents the catalytic core particle of the 26S proteasome, which is an essential component of the ubiquitin-dependent protein degradation pathway. The 20S particle is composed of 14 different but related subunits. Two outer disks, each containing seven ␣ subunits, guard the two inner heptameric ␤ subunit rings, which contain three proteolytically active sites each (1). How substrate molecules gain access to the active sites at the inner surface of the ␤ rings is not known. It has been hypothesized that on association of the 20S particle with regulatory complexes, such as the 19S cap structure or PA28, an opening in the middle of the ␣ subunit rings is induced allowing unfolded proteins to be fed into the 20S particle.Probably as a consequence of this closed structure, very few proteins have been found to be degraded by 20S proteasomes in vitro, and only very few individual fragments and cleavage sites generated during the degradation of proteins by eukaryotic 20S proteasomes have been analyzed until now. This kind of analysis is of interest for several reasons.First, it still has to be confirmed that the specificities of the proteasomal ␤ subunits observed in experiments using peptide substrates (3-8), summarized as trypsin-like, chymotrypsin (ChT)-like, and peptidylglutamyl-peptide hydrolyzing activity, correspond to cleavages performed in intact proteins, a situation physiologically more relevant.Second, the mechanism of protein degradation by the proteasome is not known. For the archebacterial proteasome, which is a less complex structure consisting of 14 identical ␣ and ␤ subunits (9), a processive model has been proposed (2, 10). Because of the high structural homology with the archebacterial particle a similar mechanism can be expected for eukaryotic proteasomes. Furthermore, it has been postulated that the 20S pro...

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated ␤-subunits, we investigated the specificities and contributions of the different ␤-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active ␤-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with ␤5/Pre2 cleaving after hydrophobic, ␤2/Pup1 after basic, and ␤1/Pre3 after acidic residues, but with the exception that ␤2/Pup1 and ␤1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with ␤5/Pre2, ␤1/Pre3, or ␤2/ Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

The first version of PAProC (Prediction Algorithm for Proteasomal Cleavages) is now available to the general public. PAProC is a prediction tool for cleavages by human and yeast proteasomes, based on experimental cleavage data. It will be particularly useful for immunologists working on antigen processing and the prediction of major histocompatibility complex class I molecule (MHC I) ligands and cytotoxic T-lymphocyte (CTL) epitopes. Likewise, in cases in which proteasomal protein degradation has been indicated in disease, PAProC can be used to assess the general cleavability of disease-linked proteins. On its web site (http://www.paproc.de), background information and hyperlinks are provided for the user (e.g., to SYFPEITHI, the database for the prediction of MHC I ligands).

Naive T lymphocytes move efficiently in lymphoid tissues while scanning dendritic cells in search of cognate complexes of peptide in major histocompatibility molecules. However, T cell migration ceases after recognition of cognate antigen. We show here that during the initiation of antigen-specific CD8(+) T cell responses, naive CD8(+) polyclonal T cells 'preferentially' interacted in an antigen-independent way with mature dendritic cells competent to present antigen to antigen-specific CD8(+) T cells. These antigen-independent interactions required expression of the chemokine receptor CCR5 on polyclonal T cells and increased the efficiency of the induction of naive, low-precursor-frequency CD8(+) T cell responses. Thus, antigen-specific CD8(+) T cells favor the priming of naive CD8(+) T cells by promoting the CCR5-dependent recruitment of polyclonal CD8(+) T cells to mature dendritic cells.

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate ␤-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions. Cells present foreign, altered self, and self antigens to cytotoxic T lymphocytes (CTL) 1 on the cell surface in the context of major histocompatibility (MHC) class I molecules. Following recognition and activation, the CTL then initiate target cell destruction. The antigenic peptides essential for target cell recognition are generated in the cytosol by proteolytic degradation of predominantly endogenous proteins. The resulting pool of peptides is a possible source for translocation of 7-15-mers by the transporter associated with antigen processing into the lumen of the endoplasmic reticulum. Translocated peptides that fulfill the criteria for binding to the available MHC class I allelic products stabilize membrane-bound, empty MHC class I heavy chains in association with ␤ 2

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