Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes.
The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon gamma (IFN-gamma) for 3 d induces a change in the proteasome subunit composition and that the beta-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous delta-subunit. IFN-gamma stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-gamma-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-gamma induction, the naturally processed nonamer peptide that is presented by MHC class I molecules appears as a minor cleavage product. IFN-gamma activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size.
Boes
B
B
Zentrum für Moleculare Biologie, University of Heidelberg, Germany.
Hengel
H
H
Ruppert
T
T
Multhaup
G
G
Koszinowski
U H
UH
Kloetzel
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D002999:Clone Cells; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D015151:Immunoblotting; D007371:Interferon-gamma / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; C071313:LMP-2 protein; D007371:Interferon-gamma; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex