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-subu nit X of the mammalian 20 S proteasome and inhibit several of its pept idase activities, However, we demonstrate that [H-3]lactacystin treatm ent modifies all the proteasome's catalytic beta-subunits. Lactacystin and its more potent derivative beta-lactone irreversibly inhibit prot ein breakdown and the chymotryptic, tryptic, and peptidylglutamyl acti vities of purified 20 S and 26 S particles, although at different rate s, Exposure to these agents for 1 to 2 h reduced the degradation of sh ort- and long-lived proteins in four different mammalian cell lines, U nlike peptide aldehyde inhibitors, lactacystin and the beta-lactone do not inhibit lysosomal degradation of an endocytosed protein, These ag ents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect pr esentation of the peptide epitope SIINFEKL, which does not require pro teolysis for presentation. Generation of most peptides required for fo rmation of stable class I heterodimers is also inhibited, Because thes e agents inhibited protein breakdown and antigen presentation similarl y in interferon-gamma-treated cells (where proteasomes contain LMP2 an d LMP7 subunits in place of X and Y), all beta-subunits must be affect ed similarly, These findings confirm our prior conclusions that protea somes catalyze the bulk of protein breakdown in mammalian cells and ge nerate the majority of class I-bound epitopes for immune recognition.