Endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) is a member of zinc metalloproteases and plays a critical rule in the antigen-processing pathway.  It trims antigenic epitope precursors to fit into MHC-Class I molecules, which are displayed on the cell surface as antigenic peptides and trigger immune responses. The activity of ERAAP significantly affects the antigenic peptide repertoire on the cell surface and therefore can regulate the cell immune response to intracellular pathogens or immune evasion by viruses and cancer cells.  However, despite its importance, imaging strategies have not been developed that can image the activity of ERAAP.  Here we synthesized the first peptide-based fluorescent probe (Ep) that can sensitively measure ERAAP activity both in solution and in living cells. The Ep probe consists of a 10-mer peptide (KKSIINFECL) substrate that is flanked by a fluorophore (BODIPY) and a quencher (Dnp).  N-terminal cleavage of the peptide by ERAAP liberates the quencher and the fluorescence of the Ep probe gets recovered.  As a good ERAAP substrate, the Ep probe was able to detect 1 µg/mL of ERAAP in solution. And the Ep probe was able to measure ERAAP activity in cells via flow cytometry and confocal microscopy. This probe also has the potential to be used for developing cell-based high throughput screening assays to discover ERAAP inhibitors.

Highly sensitive and selective peptide-based fluorescent probe for Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing (ERAAP)

253rd National Meeting (2017)

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