ABSTRACT
Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.
Subject(s)
NF-E2-Related Factor 2 , NF-kappa B , Animals , Cell Death , Deubiquitinating Enzymes/metabolism , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Mice , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Ubiquitin/metabolism , Ubiquitin-Specific Proteases/metabolism , UbiquitinationABSTRACT
The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Immunity, Innate/drug effects , Lymphoma, Large B-Cell, Diffuse/drug therapy , Psoriasis/prevention & control , Ubiquitin-Protein Ligases/antagonists & inhibitors , A549 Cells , Animals , Anti-Inflammatory Agents/chemistry , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Disease Models, Animal , Enzyme Inhibitors/chemistry , Female , HEK293 Cells , HeLa Cells , Humans , Imiquimod , Inflammation Mediators/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Lymphoma, Large B-Cell, Diffuse/immunology , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Mice, Inbred BALB C , Molecular Structure , Psoriasis/chemically induced , Psoriasis/immunology , Psoriasis/metabolism , Signal Transduction , Structure-Activity Relationship , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/genetics , Ubiquitins/metabolismABSTRACT
Ovarian tumor domain-containing protein 1 (OTUD1), an OTU-family deubiquitinating enzyme, has been reported to be involved in cancer progression through the regulation of p53 and SMAD7. However, the precise pathophysiological functions of OTUD1 remain elusive. Here, we report the establishment of OTUD1-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method. The generated antibodies recognize the N-terminal portion (aa. 1-290) of human and mouse OTUD1 proteins. In addition, immunofluorescent staining and subcellular fractionation analyses using these antibodies indicated that OTUD1 is predominantly localized in the cytosol. Thus, these mAbs can be further used to elucidate cellular functions of OTUD1 and its involvement in processes such as cancer progression.
