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.
The main part of cytosolic protein degradation depends on the ubiquitin-proteasome system. Proteasomes degrade their substrates into small peptide fragments, some of which are translocated into the endoplasmatic reticulum and loaded onto MHC class I molecules, which are then transported to the cell surface for inspection by CTL. A reliable prediction of proteasomal cleavages in a given protein for the identification of CTL epitopes would benefit immensely from additional cleavage data for the training of prediction algorithms. To increase the knowledge about proteasomal specificity and to gain more insight into the relation of proteasomal activity and susceptibility to prion disease, we digested sheep prion protein with human constitutive and immuno-20S proteasomes. All fragments generated in the digest were quantified. Our results underline the different cleavage specificities of constitutive and immunoproteasomes and provide data for the training of prediction programs for proteasomal cleavages. Furthermore, the kinetic analysis of proteasomal digestion of two different alleles of prion protein shows that even small changes in a protein sequence can affect the overall efficiency of proteasomal processing and thus provides more insight into the possible molecular background of allelic variations and the pathogenicity of prion proteins.
The mfERG allows long-term follow-up of disease progression in retinitis pigmentosa. It does not replace, but complements psychophysical methods and could be used as an objective outcome measure in upcoming treatment studies involving patients with advanced retinal diseases.
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