title: Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors.
abstract: We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC-associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor-resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Furthermore, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome 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 generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed.
authors.affiliation: Department of Microbiology, Carter Immunology Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Luckey
authors.forename: J A
authors.initials: JA
authors.lastname: Marto
authors.forename: M
authors.initials: M
authors.lastname: Partridge
authors.forename: E
authors.initials: E
authors.lastname: Hall
authors.forename: F M
authors.initials: FM
authors.lastname: White
authors.forename: J D
authors.initials: JD
authors.lastname: Lippolis
authors.forename: J
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: D F
authors.initials: DF
authors.lastname: Hunt
authors.forename: V H
authors.initials: VH
authors.lastname: Engelhard
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000502:physiology; D000483:Alleles; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D006680:HLA Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015234:HLA-A Antigens / Q000096:biosynthesis; D015788:HLA-A1 Antigen / Q000096:biosynthesis; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens / Q000096:biosynthesis; D059949:HLA-B51 Antigen; D015795:HLA-B8 Antigen / Q000096:biosynthesis; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D015853:Cysteine Proteinase Inhibitors; D006680:HLA Antigens; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015788:HLA-A1 Antigen; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens; D059949:HLA-B51 Antigen; D015795:HLA-B8 Antigen; D015395:Histocompatibility Antigens Class I; D009097:Multienzyme Complexes; D010446:Peptide Fragments; C073119:TAP1 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex