DUBs catalyze the removal of ubiquitin from native conjugates, ubiquitin C-terminal extension peptides and linear poly-ubiquitin fusion or precursor proteins. Many DUBs have esterase and amidase activities in vitro, and ubiquitin-esters or ubiquitin-amidocoumarins (ubiquitin-AMC) have been widely used as general substrates for activity assays. These activities could be physiologically relevant for the removal of linkages that might arise in vivo from nucleophilic attack by small molecules (glutathione, spermidine) on reactive ubiquitin thiolester linkages.
DUBs are classed into two distinct families: ubiquitin C-terminal hydrolases (UCHs) and the ubiquitin-specific proteases (USPs/UBPs). UCHs are relatively small enzymes (20-30 kDa) that catalyze the removal of peptides and small molecules from the C-terminus of ubiquitin. Most UCHs cannot generate monomeric ubiquitin from protein conjugates or disassemble poly-ubiquitin chains. USPs vary greatly in size (50-300 kDa) usually with N-terminal extensions which may function in substrate recognition, subcellular localization and protein-protein interactions. USPs can process ubiquitin precursors, remove ubiquitin from protein conjugates and disassemble ubiquitin chains. Isopeptidase T (IsoT) is a USP capable of binding ubiquitin and specifically disassembles free or unanchored poly-ubiquitin chains. Most recently the JAMM isopeptidases, otubains and ataxin-3/josephin have also been identified as ubiquitin-specific proteases. There are also enzymes that specifically hydrolyze C-terminal isopeptide bonds for UBLs: SUMO-specific proteases (SENPs), NEDD8-specific proteases (COP9 signalosome) and ISG15-specific proteases (UBP43).