RESUMO
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.