Investigation of the P1' and P2' sites of IQF substrates and their selectivity toward 20S proteasome subunits.

High levels of expression and activity of the 20S proteasome have been linked to many types of pathologies, including neoplasia, autoimmune disorders, neurodegenerative diseases and many more. Moreover, distinguishing between 20S proteasome catalytic subunits is neglected, although it may provide further insight into the development of pathologies. Several approaches have been developed to detect 20S proteasome activity, one of which is internally quenched fluorescent (IQF) substrates, which currently suffer from low efficiency and sensitivity. Previous reports focused on peptides including natural amino acids; therefore, in this report, we synthesized and analyzed IQF substrates with both natural and unnatural amino acids in the P1' and P2' positions to investigate their influences on selectivity toward 20S proteasome subunits. We found that elongation of the substrate by the P1' and P2' positions increased specificity in comparison to tetrapeptides. Moreover, we were able to obtain IQF substrates for the Ch-L subunit, which was characterized by higher selectivity than formerly used tetrapeptides. These findings may further contribute to the development of novel diagnostic tools for 20S proteasome-dependent disorders.