Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors

We have studied the contributions of proteasome inhibitor-sensitive and -in sensitive proteases to the generation of class I MHC-associated peptides. T he cell surface expression of 13 different human class I MHC alleles was in hibited by as much as 90% or as little as 40% when cells were incubated wit h saturating concentrations of three different proteasome inhibitors. Inhib itor-resistant class I MHC expression was not due to TAP-independent expres sion or preexisting internal stores of peptides. Furthermore, it did not co rrelate with the amount or specificity of residual proteasome activity as d etermined in in vitro proteolysis assays and was not augmented by simultane ous incubation with multiple inhibitors. Mass spectrometry was used to dire ctly characterize the peptides expressed in the presence and absence of pro teasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be genera ted in the presence of saturating levels of proteasome inhibitors. Comparis on of the peptide-binding motifs of inhibitor-sensitive and -resistant clas s I alleles further suggested that inhibitor-resistant proteolytic activiti es display a wide diversity of cleavage specificities, including a trypsin- like activity. Sequence analysis demonstrated that inhibitor-resistant pept ides contain diverse carboxyl termini and are derived from protein substrat es dispersed throughout the cell. The possible contributions of inhibitor-r esistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles a re discussed.