NOVEL DIPEPTIDE ALDEHYDES ARE PROTEASOME INHIBITORS AND BLOCK THE MHC-I ANTIGEN-PROCESSING PATHWAY

MHC (MHC-I) molecules present peptides derived from Ag that are proces sed in the cytosol. The proteasome is a multicatalytic protease comple x that is present in the cytosol and has been implicated in cytosolic Ag processing. Novel dipeptide aldehydes were designed, synthesized, a nd demonstrated to specifically inhibit the chymotrypsin-like protease activity of isolated proteasomes, but produced relatively little inhi bition of cathepsin B, a vacuolar cysteine protease. The inhibitors we re membrane permeable and inhibited intracellular cleavage of a membra ne-permeable fluorogenic substrate of the chymotrypsin-like proteasome activity. When a model Ag, OVA, was introduced into the cytoplasm of M12.B6 murine B cells by electroporation, the proteasome inhibitors bl ocked its processing for subsequent presentation by MHC-I molecules. T he inhibitors had little effect on class II MHC processing of exogenou s Ag. The potencies of different inhibitors for blockade of MHC-I Ag p rocessing correlated directly with their potencies for inhibition of t he chymotrypsin-like proteasome activity. In contrast, conventional in hibitors of vacuolar cysteine proteases (e.g., leupeptin and benzyloxy carbonyl-Phe-Ala-CHN2) had little effect on MHC-I processing or the ch ymotrypsin-like activity of isolated proteasomes. These results direct ly demonstrate that inhibition of proteasome activity blocks MHC-I Ag processing, confirming a role for proteasomes in this pathway. Moreove r, they suggest that the chymotrypsin-like activity of the proteasome may be of major importance to the cytosolic processing of at least som e Ag.