Mechanism of unanchored polyubiquitin chain production

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Recently, it has been reported that unanchored Lys63-linked polyubiquitin chains can act as a scaffold protein linking TAK1 and TAB2 in the NF-kappaB signaling pathway. In this study, we synthesized a novel fluorogenic substrate, x3Ub(G76V)-Ub-AMC and an activity-based probe, x3Ub(G76V)-Ub-VS, which enable detection of the unanchored polyubiquitin chain-liberating activity by a native chemical ligation strategy. We showed that the activity profile for the x3Ub(G76V)-Ub-AMC hydrolysis in MonoQ fractions of HeLa cell extract was different from those for Ub-AMC and Ub-GrB hydrolysis, suggesting a possibility of the substrate for endo-cleavage type deubiquitinating enzymes. We also revealed that the x3Ub(G76V)-Ub-AMC hydrolyzing activity in HT1080 cells was attenuated by tumor necrosis factor stimulation, suggesting an involvement of endo-cleavage type deubiquitinating enzymes in inflammatory responses.