Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).

Activin ligands are formed from two disulfide-linked inhibin β (Inhβ) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.