Proteasomes are self-compartmentalizing multi-subunit protease complexes performing most of the non-lysosomal proteolysis in eukaryotic cells. All proteasomes isolated from eukaryotic cells until now contain the so-called 20S proteasome as the catalytic core. In Figure 2, the crystal structure of the 20S proteasome in yeast is shown. It depicts a cylindrical particle consisting of 28 subunits arranged in four heptameric rings. The two inner ß-rings form the central cavity of the cylinder and harbor at their inner surface the proteolytic active sites. In eukaryotic 20S proteasomes, only three ß subunits (ß1, ß2 and ß5) are active, with an N-terminal threonine as the catalytic residue. Each of these subunits has a distinct substrate preference, which is usually characterized by the rate in which it cleaves small fluorogenic peptides. Intriguingly, stimulation of cells by γ-interferon causes the replacement of the three active ß-subunits ß1, ß2 and ß5 by their iso-forms ß1i, ß2i and ß5i in newly synthesized proteasomes. Because these subunits are induced by γ-interferon, signaling an infection in the vicinity of the cell, it is assumed that these new 'immuno-proteasomes' enhance the antigen procession capability of a cell. The immuno-subunits do posses a distinct cleavage preference, but it is not exactly clear how this improves antigen processing.

Figure 2: Structure of the 20S yeast proteasome published by (Groll, et al., 1997).

The α-subunits of the two outer rings form the boundary of a gated channel through which the traffic of incoming substrates and outgoing peptides is likely to proceed (Groll, et al., 2000;Kohler, et al., 2001). In vivo, the 20S core proteasome is usually found associated with 19S and / or 11S regulatory complexes. These regulators dock at the α rings and are believed to control the access to the core channel. The 19S regulators recognize proteins tagged with a poly-ubiquitin chain, which marks them for degradation. The 11S regulators are induced by γ-interferon which again makes it likely that their function enhances the antigen procession capability of a cell. In contrast to the 20S core alone, these 26S proteasomes need ATP to function.

Proteasomes are essential to life. Chromosomal deletions of each of the 14 yeast 20S proteasome genes are lethal (Heinemeyer, et al., 1991;Hilt and Wolf, 1995). Functional integrity of proteasomes has been demonstrated to be indispensable for a variety of cellular functions besides generation of antigenic peptides such as metabolic adaptation, cell differentiation, cell-cycle control, stress response and removal of abnormal proteins (Hilt and Wolf, 1996). The role as supplier of antigenic peptides was presumably taken over by the proteasome during the evolution of the immune system because of its ancient property to cleave substrates into smaller peptides (Niedermann, et al., 1997).

Originally it was thought that peptides generated by the proteasome during normal protein turn-over would be the only source of fragments for the MHC-I pathway. However it has been known for some time that around 40% of proteins are degraded by the proteasome within a minute of synthesis, which is thought to be a consequence of their inability to fold. Degradation of these defective ribosomal proteins (DRiPs) has been found to be a main source of antigenic peptides (Schubert, et al., 2000). This gives the immune system access to all proteins at the point of synthesis, independent of their lifetime and final location in the cell.

Apart from the proteasome, several other proteases have been implicated in the generation of antigenic peptides. Among these are the tripeptidyl peptidase II, furin and the thimet oligopeptidase. (Schwarz, et al., 2000). Their importance is not yet completely clear, but it can be assumed that because of their selective specificity, they can only play a role in the generation of a minority of observed antigenic peptides.

TAP is a heterodimer consisting of TAP1 and TAP2, each of which contains transmembrane domains and an ATP binding motif. No crystal structure of TAP is currently available, but it is known from sequence homology analysis that TAP belongs to the super family of ATP-binding cassette transporters (ABC transporters). The TAP genes are coded in the MHC-II locus, and are up regulated after stimulation with γ-interferon (Ayalon, et al., 1998), which implicates the role of TAP in antigen procession.

The initial selective step of TAP transport is binding of the peptide, which involves both subunits of TAP. This is followed by a slow structural reorganization of the molecule, which is believed to trigger ATP hydrolysis and peptide translocation across the membrane (Neumann and Tampe, 1999). TAP specificity has been analyzed using combinatorial peptide libraries (Uebel, et al., 1995) showing that the C-terminus and the three N-terminal residues of a peptide contribute most to binding to TAP. The optimal lengths of peptides for transport is 8-16 amino acids, but oligopeptides up to 40 residues in length have been shown to be transported (Momburg, et al., 1994;van Endert, et al., 1994).

Figure 3: Epitope (blue) in the binding groove of the MHC-I α-chain (orange). Structure published by (Khan, et al., 2000).

Loaded MHC-I molecules are heterotrimers consisting of the presented epitope bound to the polymorphic α-chain which is again bound to the invariant β2 microglobulin. Figure 3 depicts a peptide in the binding pocket of the MHC-I molecule. Polymorphism in the α chain primarily involves residues in the binding pocket, giving rise to the large variety of binding specificities observed for different MHC-I alleles. Each human has up to six different MHC-I alleles, out of 980 different ones currently known (February 2003, www3.ebi.ac.uk/Services/imgt/hla/cgi-bin/statistics.cgi).

The assembled empty MHC-I molecules are associated with TAP, and the molecule tapasin acts as a bridge between the two. This places the empty MHC-I molecules close to the peptide source and retains them there until they are loaded with a peptide. The loaded MHC-I molecules leave the ER via the Golgi apparatus and the trans-Golgi network to the cell surface. Several hundred thousand copies of MHC-I molecules each containing a single epitope are presented at any time on the cell surface, where their epitopes are scanned by CTL cell receptors as shown in Figure 4.

Figure 4: MHC-I bound epitope is scanned by T-cell receptor. Structure published in (Garboczi, et al., 1996).