Identification and characterization of DEN1, a deneddylase of the ULP family

To identify deneddylases, proteases with specificity for hydrolysis of Nedd8 derivatives, a facile method was developed for the synthesis of Nedd8 amidomethylcoumarin (a substrate) and Nedd8 vinyl sulfone (an inhibitor). Deneddylase activity is necessary to reverse the conjugation of Nedd8 to cullin, a modification that regulates at least some ubiquitin ligases. The reaction of Nedd8 vinyl sulfone with L-M(TK^-) mouse fibroblast lysates identified two deneddylases. The deubiquitinating enzyme UCH-L3 is labeled by both ubiquitin vinyl sulfone and Nedd8 vinyl sulfone. In contrast, a second and more selective enzyme is labeled only by Nedd8 vinyl sulfone. This protein, DEN1, is a 221-amino acid thiol protease that is encoded by an open reading frame previously annotated as SENP8. Recombinant human DEN1 shows significant specificity for Nedd8 and catalyzes the hydrolysis of Nedd8 amidomethylcoumarin with a K_m of 51 nM and a k_cat of 7 s^-1. The catalytic efficiency of DEN1 acting upon ubiquitin amidomethylcoumarin is 6 x 10^-4 that of Nedd8 amidomethylcoumarin and its activity on SUMO-1 amidomethylcoumarin is undetectable. This selectivity was unexpected as DEN1 is most closely related to enzymes that catalyze desumoylation. This observation expands to four the number of DUB families with members that can process the C terminus of Nedd8.