Abstract
Met1 type ubiquitination and deubiquitination are involved in the regulation of many fundamental processes such as inflammation and innate immunity, and their interference by pathogens can suppress immune responses in human cells. However, no plant-derived deubiquitinases (DUBs) against Met1 ubiquitin chains have been reported. Using a dehydroalanine (DHA)-bearing Met1 diubiquitin (Met1-diUb) suicide probe, synthesized in one-pot, we identified OTUB1 from Oryza sativa (OsOTUB1) and uncovered its preference for Met1 ubiquitin chains. Also, by resolving the apo structure of OsOTUB1 and its complex with Ub or Met1-diUb, we demonstrated that OsOTUB1 hydrolyses Met1 ubiquitin chains by activation of both the distal and proximal ubiquitin, which is different from OTULIN and expands our mechanistic understanding of the DUB-mediated hydrolysis of Met1 ubiquitin chains. Through large-scale sequence alignment and hydrolysis experiments, two sites in the S1' pocket of the OTUB subfamily (OTUBs) were found to determine the hydrolytic ability of OTUBs against Met1 ubiquitin chains, regardless of species. Furthermore, by analyzing the species distribution of OTUBs capable of hydrolyzing Met1 ubiquitin chains, we found that whereas this activity does not exist in metazoans, it is conserved in green plants (Viridiplantae). This discovery may inform studies of the differentiation between primitive plants and animals.
Activity based Deubiquitinase profiling reveals OTUB1 from oryza sativa Hydrolyzes Linear Ubiquitin Chains by Bi-modal Activation