Lactacystin and clasto-lactacystin beta-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation.
The antibiotic lactacystin was reported to covalently modify beta-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome's catalytic beta-subunits. Lactacystin and its more potent derivative beta-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the beta-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-gamma-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all beta-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition.
Craiu
A
A
Division of Lymphocyte Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Gaczynska
M
M
Akopian
T
T
Gramm
C F
CF
Fenteany
G
G
Goldberg
A L
AL
Rock
K L
KL
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017951:Antigen Presentation / Q000187:drug effects*; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007783:Lactones / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D015395:Histocompatibility Antigens Class I; D007783:Lactones; D009097:Multienzyme Complexes; D047090:beta-Lactams; C067713:lactacystin; C098869:clasto-lactacystin beta-lactone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine