title: 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 Km of 51 nm and a kcat of7s-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.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Tudeviin
authors.initials: T
authors.lastname: Gan-Erdene
authors.forename: Kolli
authors.initials: K
authors.lastname: Nagamalleswari
authors.forename: Luming
authors.initials: L
authors.lastname: Yin
authors.forename: Kenneth
authors.initials: K
authors.lastname: Wu
authors.forename: Zhen-Qiang
authors.initials: ZQ
authors.lastname: Pan
authors.forename: Keith D
authors.initials: KD
authors.lastname: Wilkinson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D000075687:NEDD8 Protein; D009842:Oligopeptides; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism; D013379:Substrate Specificity; D013450:Sulfones / Q000737:chemistry; D013869:Thiolester Hydrolases / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C513172:Nedd8 protein, mouse; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D013450:Sulfones; D025801:Ubiquitin; D014452:Ubiquitins; C009873:divinyl sulfone; C085706:FLAG peptide; D013869:Thiolester Hydrolases; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; C477008:SENP8 protein, human