Global evaluation of the role of ubiquitin in MHC Class I-restricted antigen processing. (106.35)

Abstract

Abstract The multicatalytic proteasome plays an indispensable role in cellular proteolysis and in the production of most MHC class I-restricted antigenic peptides. Although ubiquitinylation was originally considered to be necessary for directing substrates to proteasomal destruction, growing evidence supports a role for ubiquitin-independent degradation as a substantial portion of intracellular proteolysis. To explore the importance of ubiquitin in MHC class I-restricted antigen processing, we have employed an inducible vaccinia virus system to overexpress wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells. We have previously shown that abrogation of polyubiquitination in this manner inhibited epitope presentation from endoplasmic reticulum-targeted model antigens but had little impact on antigen presentation from cytosolic proteins. We are now seeking to elucidate the selective role of ubiquitin in MHC class I antigen processing by global evaluation of MHC class I-associated peptide display in the presence or absence of polyubiquitination, using mass spectrometric identification of peptides and comparative analysis of parent protein families.