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 -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 wit...

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Bibliographic Details
Published in:The Journal of immunology (1950) 2001-08, Vol.167 (3), p.1212-1221
Main Authors: Luckey, Chance John, Marto, Jarrod A, Partridge, Megan, Hall, Ed, White, Forest M, Lippolis, John D, Shabanowitz, Jeffrey, Hunt, Donald F, Engelhard, Victor H
Format: Article
Language:English
Subjects:
Alleles
ATP Binding Cassette Transporter, Subfamily B, Member 2
ATP-Binding Cassette Transporters - physiology
Cell Line, Transformed
Cysteine Endopeptidases - metabolism
Cysteine Proteinase Inhibitors - pharmacology
Flow Cytometry
Histocompatibility Antigens Class I - biosynthesis
Histocompatibility Antigens Class I - genetics
Histocompatibility Antigens Class I - metabolism
HLA Antigens - biosynthesis
HLA Antigens - genetics
HLA Antigens - metabolism
HLA-A Antigens - biosynthesis
HLA-A1 Antigen - biosynthesis
HLA-A2 Antigen
HLA-B Antigens - biosynthesis
HLA-B51 Antigen
HLA-B8 Antigen - biosynthesis
Humans
Mass Spectrometry
Multienzyme Complexes - antagonists & inhibitors
Multienzyme Complexes - metabolism
Peptide Fragments - biosynthesis
Peptide Fragments - genetics
Peptide Fragments - metabolism
Proteasome Endopeptidase Complex
proteasome inhibitors
Substrate Specificity - immunology
Transfection
Tumor Cells, Cultured
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Summary: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.
ISSN:0022-1767
1550-6606
DOI:10.4049/jimmunol.167.3.1212