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.

The DDB1- and CUL4-associated factor (DCAF) family functions as substrate receptors within Cullin4-really interesting new gene (RING) ubiquitin ligases (CRL4s), facilitating proteasomal degradation of targeted substrates. Although CRL4-based targeted protein degradation (TPD) has emerged as a promising strategy to modulate undruggable proteins, the complex formation, substrates, and functional properties of many DCAFs remain poorly defined. In this study, using proximity biotinylation-based interactome analysis in human HEK293T cells, we systematically annotated interactors and functional associations of individual DCAFs. Furthermore, we identified substrates of the model DCAFs COP1 and DCAF3 using proximity biotinylation coupled with multi-omics approaches. By combining biochemical and cell-based analyses, we establish CRL4 complex formation and DCAF autodegradation as experimentally tractable proxy indicators to evaluate DCAF degradation activity and propose a set of high-activity DCAFs. These datasets establish a resource for functional characterization of DCAFs and provide a framework for their prioritization in TPD.

Thalidomide, a small-molecule drug, caused a global drug disaster more than half a century ago. Thalidomide derivatives are widely used to treat several hematological cancers. Recent studies have revealed that thalidomide and its derivatives act as protein degraders that bind to cereblon (CRBN), the substrate-recognition receptor of a complex-type E3 ubiquitin ligase, and degrade neo-substrates that are not their original substrates. Furthermore, they are currently being used in proteolysis-targeting chimeras (PROTACs), which are chimeric protein degraders. Protein degraders open up a new mechanism of drug action and proteolysis and have the potential to target various therapeutic proteins, including undruggable proteins. Analysis of drug-dependent protein-protein interactions (PPIs) is crucial for the development and use of this new class of protein degraders. We developed an in vitro and intracellular protein degrader-dependent PPI analysis method using a cell-free protein synthesis system and proximity-dependent biotinylation. Using these analytical methods, we successfully identified several neo-substrates involved in the mechanism of action of thalidomide. Furthermore, we developed thalidomide derivatives and PROTACs with enhanced selectivity for neo-substrates or target proteins involved in the anti-hematological cancer effects. In this paper, we review the history of thalidomide and the research achievements related to protein degraders, as represented by thalidomide derivatives.