Identification and Characterization of DEN1, a Deneddylase of the ULP Family

Abstract

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 ؋ 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. Ubiquitin (Ub)1 and the ubiquitin-like proteins (Ubl) constitute a family of proteins that are post-translationally conjugated to other proteins to target them for specific localization (1-4). Thus, attachment of a Lys 48 -linked polyubiquitin chain targets proteins to the proteasome, the multicatalytic proteinase that is responsible for most regulated intracellular proteolysis (5, 6). Ubiquitination is also involved in numerous other processes, including DNA repair, establishment, and maintenance of chromatin structure, receptor internalization, and sorting, and modulation of signal transduction pathways (5, 7). All ubiquitin-like proteins are activated by evolutionarily related E1 enzymes, and passed on (as thiol esters) to a family of E2 conjugating enzymes. In some cases this E2-ubiquitin thiol ester can directly catalyze ubiquitination, whereas in most cases a third enzyme, the ubiquitin ligase, is required to conjugate ubiquitin (and ubiquitin-like proteins) to the target protein (7).Several ubiquitin-like proteins are also conjugated to other proteins and likewise act as targeting signals for the attached proteins (8,9). Nedd8 is about 50% identical to ubiquitin and its conjugation to the cullin component of several ubiquitin ligases is thought to increase the efficiency of polyubiquitination (4). SUMO-1, -2, and -3 are closely related proteins with about 20% identity to ubiquitin that are attached to numerous proteins in the nucleus and to septins that accumulate at the bud neck of yeast (2, 10 -12). Whereas the precise sites of localization and the mechanisms by which it is accomplished are not known, several important cellular regulators are sumoylated. These include p5...

Total Chemical Synthesis of SUMO and SUMO‐Based Probes for Profiling the Activity of SUMO‐Specific Proteases

“…As expected, SDS‐PAGE analysis showed diSUMO‐reacted bands for all SENPs except SENP8, which is reported to be a NEDD8‐specific protease26 (Figure S6). Next, we tested the reactivity of the probe towards known SUMO proteases in cell lysates.…”

Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

“…SENP7 is not characterized in the literature to date. Unlike the other characterized SENPs, SENP8 removes Nedd8 (another ubiquitin-like protein) from target proteins (11). So far, five ULP1 genes have been identified in Arabidopsis thaliana.…”

Transcriptional ERRγ2-mediated activation is regulated by sentrin-specific proteases

“…Both, superactivation by replacing the potential phosphorylation acceptor Ser 45 with alanine and a phosphorylation-dependent mobility shift of protein DNA complexes during gel electrophoresis speak in favor of a functional PDSM. Generating an acidic moiety, by replacing both Ser 44 and Ser 45 with glutamic acid, results in down-regulation, supporting the view that the PDSM is the regulated form of an NDSM. This is further corroborated by the repression effect of phosphomimetic mutants in the downstream region.…”

“…demonstrates activity only against Nedd8-modified proteins [44][45][46], it is generally assumed that all other SENPs cleave SUMOs. However, this has only been investigated in detail with SENP1, 2, 3 and 5.…”