Decoding the Linkage Specificity of Deubiquitinating Enzymes with Di- and Tetraubiquitin: Are Longer Polyubiquitin Chains the Key?

Nate Russell, Steven Feudo, Zac Stiffler, Jarad Yost, Greg Costakes, Carsten Schwerdtfeger, and Bradley Brasher


Deubiquitinating enzymes (DUBs) play an essential role in many cellular processes and their dysfunction results in a number of human diseases. Despite this important role, the substrate specificity and kinetic parameters of many DUBs are poorly defined. Monoubiquitin based fluorescent substrates such as Ubiquitin-AMC (Ub-AMC) have been quite useful for DUB characterization, though not all DUBs utilize Ub-AMC well. The use of monoubiquitin substrates yields an incomplete picture of the preferred substrates and kinetic activity of DUBs as UbAMC lacks the extended structure and isopeptide bonds that polyubiquitin (polyUb) chains possess. Additionally, a paucity of full-length enzymes and longer (>2 Ub) polyUb chains of most linkage types has made it challenging to determine the types of polyUb each DUB can process. Here we provide an analysis of 36 DUBs (mostly full-length) for linkage specificity and possible cleavage mechanisms against a complete set of all 8 linkages of di- and tetraubiquitin (except K27-linked tetraubiquitin).