Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses.
Here we describe a general method to synthesize and use a panel of reagents with selectivity for deubiquitinating enzymes exhibiting specificity for each ubiquitin-like protein. A substrate (Ubl-AMC), a reversible inhibitor (Ubl-aldehyde), and an active-site-directed irreversible inhibitor (Ubl-vinylsulfone) are described for each Ubl. Because of space constraints, we give details for only the Nedd8 derivatives, but these methods have been used in our laboratory to produce these derivatives of Ubiquitin, Nedd8, Sumo-1, Sumo-2, and Isg15. These reagents are useful in defining the specificity of DUBs, as well as in purifying and identifying these important proteins. The reagents are selective and useful in crude extracts. The reactivity of these reagents reveals differences in both the S1 and S1' sites of deubiquitinating enzymes. Only active enzymes are efficiently detected with these reagents. Published results indicate that specificity is not strictly defined by the evolutionary relationships of these DUBs.
Wilkinson
Keith D
KD
Department of Biochemistry, Emory University, Atlanta, Georgia, USA.
Gan-Erdene
Tudeviin
T
Kolli
Nagamalleswari
N
D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006868:Hydrolysis; D047668:Inteins; D000075687:NEDD8 Protein; D012326:RNA Splicing; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
D017931:DNA Primers; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D025801:Ubiquitin; D014452:Ubiquitins