Abstract
The multicatalytic proteinase complex (MPC, proteasome) is assembled from 14 nonidentical protein subunits. It expresses five distinct proteolytic activities, including a chymotrypsin-like activity, cleaving after hydrophobic residues, and a branched chain amino acid-preferring component (BrAAP), cleaving preferentially after branched chain residues. Exposure of cells to interferons leads to replacement of the X, Y, and Z subunits by the LMP2, LMP7, and MECL1 subunits. This "immunoproteasome" is critical to processing of certain antigens. The enzymatic basis for enhanced antigen processing has not been determined. To gain insight into this question, we examined sites and relative rates of cleavage of bonds in denatured, reduced, carboxyamidomethylated lysozyme, a 129-amino acid protein, by MPC from bovine spleen, in which the X, Y, and Z subunits are replaced by LMP2, LMP7, and MECL1. We compared cleavages to those catalyzed by MPC from bovine pituitary, which contains only the X, Y, and Z subunits. We found marked increases in the rates and number of cleavages after branched chain residues in reduced, carboxyamidomethylated lysozyme by the spleen MPC. This was largely due to accelerated cleavages of bonds after a Phi-X-Br motif, where Phi is a hydrophobic residue, X is a small neutral or polar residue, and Br is a branched chain residue. Inhibitors with these structural properties were selective and potent inhibitors of the BrAAP activity of the spleen MPC. The above findings indicate that alterations in activity and substrate specificity of the BrAAP activity are important factors underlying the altered cleavages after hydrophobic residues associated with incorporation of interferon-inducible subunits. The potential relevance of the findings to antigen processing functions of MPC is discussed.
Highlights
Tous, intracellular, proteolytic particle that is essential for survival of eukaryotic cells
Exposure of cells to interferon-␥ results in expression of LMP2 and LMP7, the two subunits encoded by genes in the major histocompatibility complex (MHC) class II gene region, and of a third interferon-␥inducible gene product, MECL1 [13,14,15,16, 21, 22], called LMP10
The multicatalytic proteinase complex (MPC) is involved in processing most peptide antigens presented on MHC class I molecules [17, 38]
Summary
Tous, intracellular, proteolytic particle that is essential for survival of eukaryotic cells (for reviews see Refs. 2–5). Identification of genes for two interferon-␥-inducible MPC subunits within the major histocompatibility complex (MHC) class II gene region [13,14,15,16] and subsequent experiments showing that MPC inhibitors block class I antigen presentation [17, 18], suggest that the MPC is a major factor involved in processing cytoplasmic and nuclear proteins into antigenic peptides These 8- or 9-amino acid peptides are transported into the endoplasmic reticulum where they are bound to major histocompatibility complex class I molecules [19, 20]. The form of MPC containing the interferon-␥-inducible subunits has been referred
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