The first version of the major histocompatibility complex (MHC) databank SYFPEITHI: database for MHC ligands and peptide motifs, is now available to the general public. It contains a collection of MHC class I and class II ligands and peptide motifs of humans and other species, such as apes, cattle, chicken, and mouse, for example, and is continuously updated. All motifs currently available are accessible as individual entries. Searches for MHC alleles, MHC motifs, natural ligands, T-cell epitopes, source proteins/organisms and references are possible. Hyperlinks to the EMBL and PubMed databases are included. In addition, ligand predictions are available for a number of MHC allelic products. The database content is restricted to published data only.

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.

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70-and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitinconjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70⅐BAG-1⅐CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitinlike domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.The control and maintenance of the three-dimensional structure of proteins are prerequisites for cell survival and involve a cooperation of molecular chaperones and energy-dependent proteases (1-4). Molecular chaperones recognize hydrophobic regions exposed on unfolded proteins and stabilize non-native conformations. As a consequence formation of insoluble protein aggregates is prevented, and folding to the native state is promoted. On the other hand, energy-dependent proteases, such as the eukaryotic 26 S proteasome, degrade irreversibly damaged proteins that fail to be folded properly.Selection of proteins for degradation by the proteasome involves ubiquitin conjugation (5-7). A polyubiquitin chain is attached to a protein substrate through the concerted action of a ubiquitin-activating enzyme (E1), 1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein isopeptide ligase (E3). In contrast to the presence of only one type of E1 enzyme in the eukaryotic cytosol, E2 and E3 enzymes are recruited from large protein families and mediate a specific recognition of a large repertoire of protein substrates. A polyubiquitin chain generated through the linkage of lysine 48 residues of successive ubiquitin moieties is usually sufficient to target a protein substrate to the proteasome, where finally deubiquitylation, unfolding, and degradation occur.Recent studies shed light onto molecular mechanisms underlying the cooperation of molecular chaperones with the ubiquitin/proteasome system during protein quality control. Two co-chaperones, CHIP and BAG-1, are of central importance in this regard. The CHIP protein was shown to act as a ...

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

There is substantial need for molecularly defined tumor antigens to prime cytotoxic T cells in vivo for cancer immunotherapy, especially in the case of tumor entities for which only a few tumor antigens have been defined so far. In this review, we present the "Tübingen approach" to identify, select, and validate large numbers of MHC/HLA class I-associated peptides derived from tumor-associated antigens. Step 1 is the identification of naturally presented HLA-associated peptides directly from primary tumor cells. Step 2 is selection of tumor-associated peptides from step 1 by differential gene expression analysis and data mining. Step 3 is validation of selected candidates by monitoring in vivo T-cell responses in the context of patient-individualized immunizations. Our approach combines methods from genomics, proteomics, bioinformatics, and T-cell immunology. The aim is to develop effective immunotherapeutics consisting of multiple tumor-associated epitopes in order to induce a broad and specific immune response against cancer cells.

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Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8 -11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K b -restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODCLEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K b and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides. MHC1 class I molecules associate with peptides of 8 -11 amino acids derived from the proteolytic degradation of intracellular protein antigens and present them to CD8ϩ T cells on the cell surface (1-3). Although the vast majority of class I ligands are translocated from the cytosol into the endoplasmic reticulum (ER) by the ATP-dependent transporter associated with antigen processing (TAP) (3), alternative pathways have been described including the liberation of minigene-encoded peptides from ER signal sequences (4 -6), TAP-independent processing of signal sequences (7,8), and processing of membrane-associated or soluble proteins in the secretory pathway (9 -12). Peptides that are not retained in the ER by binding to class I or glycosylation are released back into the cytosol through the Sec61 channel (13-15). Studies using membrane-permeable inhibitors of proteasomes have indicated that the proteasome is the major proteolytic activity responsible for the generation of antigenic peptides (16 -21), although the incomplete inhibition of antigen processing by proteasome inhibitors has also demonstrated the involvement of nonproteasomal cytosolic proteases (22-28).The proteasome is an abundant cytosolic multisubunit protease consisting of the proteolytic 20 S core particle that associates with PA700 regulatory complexes to form 26 S proteasomes or with PA28 comp...

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