A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors

Abstract

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

“…OTUD1 primarily cleaves K63-linked diubiquitin [ 11 ], but it also cleaves K6-, K11- and K48-chains [ 13 , 14 ]. Furthermore, OTUD1 reportedly cleaves K33-chains in SMAD7 [ 15 ], and atypical K6-, K11-, and K29-chains from IRF3 [ 16 ].…”

OTUD1 deubiquitylase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

“…OTUD1 primarily cleaves K63-linked diubiquitin 17 , but it also cleaves K11-and K48-chains at higher concentrations 29 . The DUB protein array analysis showed that OTUD1 preferentially cleaves the K63-ubiquitin chain, followed by the K6-and K48-chains 30 . However, several cellular analyses suggested that OTUD1 cleaves K33-linked polyubiquitin chains in SMAD7 18 , and atypical K6-, K11-, and K29-linked polyubiquitin chains from IRF3 21 .…”

The Ubiquitin Interacting Motif-Like Domain of Met4 Selectively Binds K48 Polyubiquitin Chains

“…K48 tetra-ubiquitin chains were incubated with either UIMLx2, SMT3, or buffer for several minutes before adding the deubiquitylating enzyme. Two DUBs were used, the K48 ubiquitin chain specific OTUB1 and the less specific USP2 (catalytic domain) (46)(47)(48)(49), to cleave the K48 tetra-ubiquitin chains. OTUB1 was able to process the K48 tetra-ubiquitin chains after 30 mins (Figure 5D) and after 120 mins a majority is cleaved to mono-ubiquitin.…”