The catabolic mechanism in cells that makes the initial cleavages to generate most presented peptides is the proteasome. Since the proteasome and its connection to antigen presentation has been well described elsewhere, we will cover this subject here relatively briefly and provide readers with references to representatives and/or recent papers and reviews.
The proteasome is a large barrel-shaped particle present in the cytosol and nucleus of all eukaryotic cells (3
) that is responsible for degrading of a majority of cellular proteins (6
). At its core is a 20S cylinder that is composed of two outer alpha rings and two inner beta rings with a central channel into which protein substrates enter and are cleaved (3
). The beta ring contains three active sites, each of which is formed by a different subunit: B1, B2 and B5 (3
). All three of these subunits work through the same catalytic mechanism wherein an N-terminal threonine residue makes a nucleophilic attack on a peptide bond of the substrate. However the three active sites each have different specificities, cleaving preferentially on the carboxylic side of either hydrophobic residues (B5), basic residues (B1), or acidic ones (B2) (3
The strongest evidence that the proteasome plays a major role in the generation of most presented peptides comes from studies using highly specific inhibitors of the proteasome’s threonine-active sites. In living cells, these agents block completely the presentation of peptides from antigens that require proteolysis, but have no effect on ones that do not need cleavage (e.g. when epitopes are expressed directly from minigenes) and markedly limit the overall supply of peptides to MHC class I molecules (6
While the immune system has utilized this phylogentically older pathway for a source of peptides it also evolved modifications that are thought to play a special role in antigen presentation. One modification is an alternate set of active site subunits (B1i/LMP2, B2i/MECL1, B5i/LMP7) that when expressed preferentially incorporate into newly assembling proteasomes in place of the “standard” B1, B2 and B5 subunits to form so called “immunoproteasomes” (3
). These are constitutively expressed in dendritic cells, lymphocytes, and thymic epithelium and are induced in all cells by proinflammatory cytokines such as IFN-γ.
The net effect of changing these beta subunits is to alter the catalytic activity of the proteasome’s active sites. This changes the cleavages made in protein substrates both quantitatively and qualitatively and thereby results in the production of a different set of peptides (7
). Where it has been examined for individual antigens, immunoproteasomes sometimes make more or less of a particular epitope (3
). It has been predicted that immunoproteasomes generate peptides that are more favorable for antigen presentation, however, how often this is the case and how important immunoproteasomes are to MHC class I antigen presentation is not fully resolved (6
). Mice that lack one or two of the immunoproteasome subunits have defects in the presentation of selected epitopes and in some cases partial reductions in the generation of presented peptides overall (7
). However, a more complete understanding of the contribution of immunoproteasomes to antigen presentation and maybe even to nonimmune functions awaits the generation of mice that lack all three inducible subunits.
Thymic medullary epithelium also constitutively express another B5 subunit (B5t) that preferentially incorporates with B1i plus B2i into proteasomes called “thymoproteasomes” (9
). Mice that lack either B2i or especially B5t show marked reductions in the generation of CD8 T cells and defects in the T cell repertoire, presumably because changes in the peptide repertoire lead to altered T cell selection in the thymus.
Another apparent immune modification of the proteasome is the PA28 complex that is constitutively expressed in dendritic cells and is induced in other cells by IFN-γ. PA28 is a heterohexameric (α3β3) ring that binds to one or both ends of the 20S proteasome. Upon binding it increases the catalytic activity of all three of the proteasome’s active sites (3
) and may do so at least in part opening the ends of the 20S particle allowing substrates to enter this particle. This change in activity can lead to altered cleavages in substrates (10
) and increases or decreases in the generation of at least some MHC class I-presented peptides by purified proteasomes (11
) Similarly, transfection of PA28 into cells enhanced the presentation of some but not all epitopes (3
) and increased the surface expression of some MHC class I molecules while decreasing the levels of other ones (12
) Consistent with these findings PA28 −/−
mice have a defect in presenting some antigens but not others (13
So called “hybrid” proteasomes can form with PA28 on one end and a PA700 complex on the other. PA700 confers on the proteasome complex the ability to degrade polyubiquitinated substrates (10
). Where examined “hybrid” proteasome particles make up ~20% of proteasomes in cells (14
). Whether PA28 exerts its effects on antigen presentation through proteasomes with or without PA700 is presently not clear.