Generation of MHC class I-presented peptides in cells over-expressing a dominant interfering ubiquitin (B43)

Abstract

Abstract The participation of the proteasome in MHC class I-restricted antigen processing is well documented, but the degree to which targeting of antigenic proteins to this multicatalytic protease is ubiquitin-dependent remains unclear. To address this question, we over-expressed wild type ubiquitin (Ub-WT) and lysineless ubiquitin (Ub-K0) in mammalian cells using an inducible vaccinia system. Compared to Ub-WT, the Ub-K0 potently abrogated the conjugation of ubiquitin to protein substrates, and subsequently attenuated total protein degradation. In co-infection assays, the Ub-K0 significantly inhibited specific epitope presentation from cytosolic N-end Rule substrates and an exocytic model antigen, but had little effect on antigen presentation from a stable cytosolic antigen. Interestingly, the inhibitory effect on antigen presentation by Ub-K0 decreased when the exocytic model antigen is directed to the cytoplasm, and increased when the stable cytosolic antigen is directed to the endoplasmic reticulum (ER). Despite similar levels of surface MHC class I molecules, different peptide profiles bound to surface class I molecules were observed from cells over-expressing Ub-WT and Ub-K0. The results suggest that cytosolic proteins and glycoproteins may be targeted to proteasome through different pathways to generate MHC class I peptides. This study is supported by NIH grant AI39501.