Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α

► Substrates of prolyl oligopeptidase (POP) and fibroblast activation protein α (FAP). ► Bias against charged residues at the P1' and P2' positions for POP-specific substrates. ► FAP-specific substrates preferred charged residues at the P1' and P2' positions. ► No serine or threonine residues at P1' and P2' positions for FAP-specific substrates. ► In synthetic format, some physiologic substrates of FAP are also cleaved by POP.