Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 31, Pages 28892-28900Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M302890200
Keywords
-
Categories
Funding
- FIC NIH HHS [F05 TW05461] Funding Source: Medline
- NIGMS NIH HHS [GM066355, GM061051, GM30308] Funding Source: Medline
Ask authors/readers for more resources
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 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available