RESUMO
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.
Assuntos
Fator 2 Relacionado a NF-E2 , NF-kappa B , Animais , Morte Celular , Enzimas Desubiquitinantes/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Imunidade Inata/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Psoríase/prevenção & controle , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Células A549 , Animais , Anti-Inflamatórios/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Células HeLa , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Psoríase/induzido quimicamente , Psoríase/imunologia , Psoríase/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
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.