SUBSTRATE-BINDING AND SEQUENCE PREFERENCE OF THE PROTEASOME REVEALED BY ACTIVE-SITE-DIRECTED AFFINITY PROBES

Background: The proteasome is a multicatalytic protease complex respon sible for most cytosolic protein breakdown. The complex has several di stinct proteolytic activities that are defined by the preference of ea ch for the carboxy terminal (P1) amino acid residue. Although mutation al studies in yeast have begun to define substrate specificities of in dividual catalytically active p subunits, little is known about the pr inciples that govern substrate hydrolysis by the proteasome. Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to st udy substrate binding and specificity of the proteasome. Removal of th e aromatic amino-terminal cap of the potent tripeptide vinyl sulfone p roteasome inhibitor oxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition . Addition of a fourth amino acid (P4) to the tri-leucine core sequenc e fully restored inhibitory potency. (125)l-labeled peptide vinyl sulf ones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activ ity, Changing the amino acid in the P4 position resulted in dramatical ly different profiles of beta-subunit modification, Conclusions: The P 4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlatio ns between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual beta subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfo nes, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These st udies indicate that it is possible to generate inhibitors that are lar gely specific for individual beta subunits of the proteasome by modula tion of the P4 and carboxy-terminal vinyl sulfone moieties.