Active-site-directed Irreversible Inhibitors and Substrates

Active-site-directed Irreversible Inhibitors and Substrates 

The most promising tools developed for this sort of analysis are active-site-directed irreversible inhibitors of DUBs. These inhibitors are ubiquitin or ubiquitin-like proteins chemically modified at the C-terminus by an electrophilic moiety such as a Michael acceptor or alkyl halide. The modified ubiquitin can be incubated with a purified DUB or a cell lysate containing DUB activity. Ubiquitin vinyl sul-fone (UbVS) is one such irreversible inhibitor because the vinyl sulfone moiety reacts with the active-site cysteine of the DUB, forming a thioether linkage. The covalent adduct is stable and can be detected in a variety of ways. Labeling of DUBs is specific, as only a DUB active-site cysteine will efficiently react with the vinyl sulfone moiety. 

This labeling has been used with success in yeast where 6 of the 17 known DUBs were labeled with UbVS [115]. Incomplete labeling likely results from DUBs that do not act on mono-ubiquitin or where the UbVS could not access the active site. The labeling has also been used with great success in mammalian cell lysates to identify novel ubiquitin DUBs [41]. A novel deneddylating enzyme and a novel DUB that acts on autophagy-related UbL proteins have also been identified using vinyl sulfone labeled probes [26, 37, 116]. 

This ubiquitin intein system can also be utilized to make a DUB substrate rather than inhibitors by attaching a C-terminal fluorescent tag such as 7-amidomethylcoumarin (AMC) instead of vinyl sulfone. DUBs cleave the ubiquitin derivative and release the fluorescent tag, a process that can be followed fluoromet-rically. Eluorometric assays can then be used to determine a particular DUB s preferred substrate or to quantitate DUB activity in crude lysates. AMC substrates 

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