The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate

Intracellular protein degradation is a major source of short antigenic pept ides that can be presented on the cell surface in the context of major hist ocompatibility class I molecules for recognition by cytotoxic T lymphocytes . The capacity of the most important cytosolic protease, the 20 S proteasom e, to generate peptide fragments with an average length of 7-8 amino acid r esidues has been thoroughly investigated. It has been shown that the cleava ge products are not randomly generated, but originate from the commitment o f the catalytically active subunits to complex recognition motifs in the pr imary amino acid sequence. The role of the even larger 26 S proteasome is l ess well defined, however. It has been demonstrated that the 26 S proteasom e can bind and degrade ubiquitin-tagged proteins and minigene translation p roducts in vivo and in vitro, but the nature of the degradation products re mains elusive. In this study, we present the first analysis of cleavage pro ducts from in vitro digestion of the unmodified model substrate beta-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 substanti ally different, sets of fragments by following very similar instructions.