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.
Highlights
Protein ubiquitination is a posttranslational protein modification involved in the regulation of numerous biological processes, such as cell growth, responses to many kinds of biotic and abiotic stresses, and pathogenesis
We evaluated the inhibitory effects of two a non-specific deubiquitinating enzymes (DUBs) inhibitor, and SJB3-019A, which preferably inhibits some DUBs, such as USP1, a non-specific
DUBs were synthesized as at least partially active forms; this is the first report of the activities and linkage specificities of such a large number of full-length DUBs
Summary
Protein ubiquitination is a posttranslational protein modification involved in the regulation of numerous biological processes, such as cell growth, responses to many kinds of biotic and abiotic stresses, and pathogenesis. In the process of ubiquitination, ubiquitin, a small protein consisting of 76 amino acids, is covalently attached to lysine (K) residues in the target protein through its Biomedicines 2020, 8, 152; doi:10.3390/biomedicines8060152 www.mdpi.com/journal/biomedicines. Additional ubiquitin is conjugated to the ubiquitin attached to the target protein or to the free ubiquitin through its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), or the initiation methionine (M1), for the formation of polyubiquitin chains. The regulation of cellular polyubiquitin chains is a key issue for understanding their biological functions. As a counteraction to polyubiquitin formation, deubiquitinating enzymes (DUBs) cleave the polyubiquitin chains or remove the ubiquitin molecules conjugated to target proteins, and act as negative regulators of ubiquitin- and polyubiquitin-mediated cellular processes [3].
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