Lactacystin and clasto-Lactacystin β-Lactone Modify Multiple Proteasome β-Subunits and Inhibit Intracellular Protein Degradation and Major Histocompatibility Complex Class I Antigen Presentation

Abstract

The antibiotic lactacystin was reported to covalently modify ␤-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [ 3 H]lactacystin treatment modifies all the proteasome's catalytic ␤-subunits. Lactacystin and its more potent derivative ␤-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 ␤-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-␥-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all ␤-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.

Covalent modification of the active site threonine of proteasomal β subunits and theEscherichia colihomolog HslV by a new class of inhibitors

“…While the predominant species modified by lactacystin is X, modification of other subunits that require higher concentrations of drug may have escaped detection due to the relatively low concentration of labeled compound used, as confirmed in subsequent work (23). We conclude that 125 I-NIP-L 3 VS labels at least five distinct ␤ subunits, all of which are also targets for lactacystin.…”

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

“…Studies using catalytic site-specific mutants of yeast proteasome and site-specific inhibitors have demonstrated that the chymotrypsin-like activity primarily determines the rate of protein degradation (Craiu et al, 1997;Lee and Goldberg, 1996). Therefore, the inhibition of this activity probably has the most significant consequences for key processes involved in cell survival.…”

“…The inner two rings contain the β subunits. Three of the β subunits (β1, β2, β5) contain the catalytic sites that perform distinct proteolytic activities (Craiu et al, 1997;Lee and Goldberg, 1996;Rock et al, 1994). These activities are classified as caspase-like (β1), trypsin-like (β2), and chymotrypsin-like (β5) as defined by cleavage after acidic, basic, or hydrophobic amino acids, respectively.…”

Antigen Targeting to CD11b Allows Efficient Presentation of CD4+ and CD8+ T Cell Epitopes and In Vivo Th1-Polarized T Cell Priming

“…Therefore, we tested whether the proteasome is required for CyaA-MalE-OVA delivery to MHC II pathway. BMDC were incubated for 1 h with lactacystin, a 20 S proteasome inhibitor (39,40), and the Ag were then added. As shown in Fig.…”