RESUMEN
The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-κB-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63-linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Receptor Toll-Like 7/metabolismo , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Citocinas/análisis , Células HEK293 , Humanos , Inmunidad Innata , Immunoblotting , Inmunoprecipitación , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , Reacción en Cadena de la Polimerasa , Quinolinas , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Reconocimiento de Patrones , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 7/agonistas , Receptores Toll-Like/agonistas , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Virus infection induces host antiviral responses including induction of type I IFNs. Transcription factor IFN regulatory factor 3 (IRF3) plays an essential role and is tightly regulated in this process. Herein we report that TRIM21 (tripartite motif-containing 21) is significantly induced and interacts with IRF3 upon RNA virus infection. Ectopic expression or knockdown of TRIM21 could respectively enhance or impair IRF3-mediated gene expression. Mechanistically, TRIM21 interferes with the interaction between Pin1 (peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1) and IRF3, thus preventing IRF3 ubiquitination and degradation. A conserved motif in the B 30.2 domain of TRIM21 is critical for its modulation of IRF3 function, while the RING finger is dispensable. Host antiviral responses are significantly boosted or crippled in the presence or absence of TRIM21. Our results identify TRIM21 as an essential modulator of IRF3 stability and demonstrate that it positively regulates the strength and duration of primary antiviral response, thus further strengthening the notion that the TRIM family is evolutionarily integrated with innate immunity.
Asunto(s)
Antivirales/metabolismo , Proteínas de Unión al ADN/fisiología , Factor 3 Regulador del Interferón/metabolismo , Proteínas Nucleares/fisiología , Infecciones por Respirovirus/virología , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Evolución Molecular , Humanos , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Factor 3 Regulador del Interferón/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/fisiología , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/metabolismo , Ribonucleoproteínas , Virus Sendai/inmunología , Ubiquitinación/inmunologíaRESUMEN
Virus infection induces host antiviral responses, including induction of type I interferons. Transcription factor interferon regulatory factor 3 (IRF3) plays a pivotal role and is tightly regulated in this process. Here, we identify HERC5 (HECT domain and RLD 5) as a specific binding protein of IRF3 by immunoprecipitation. Ectopic expression or knockdown of HERC5 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, HERC5 catalyzes the conjugation of ubiquitin-like protein ISG15 onto IRF3 (Lys193, -360, and -366), thus attenuating the interaction between Pin1 and IRF3, resulting in sustained IRF3 activation. In contrast to results for wild-type IRF3, the mutant IRF3(K193,360,366R) interacts tightly with Pin1, is highly polyubiquitinated, and becomes less stable upon Sendai virus (SeV) infection. Consistently, host antiviral responses are obviously boosted or crippled in the presence or absence of HERC5, respectively. Collectively, this study characterizes HERC5 as a positive regulator of innate antiviral responses. It sustains IRF3 activation via a novel posttranslational modification, ISGylation.
Asunto(s)
Citocinas/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ubiquitinas/metabolismo , Animales , Línea Celular , Citocinas/genética , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata/fisiología , Factor 3 Regulador del Interferón/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/genéticaRESUMEN
Intracellular RNA viruses are sensed by receptors retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) that trigger the formation of MAVS signal complex on mitochondria. Consequently, this leads to the activation of TANK-binding kinase 1 (TBK1) and phosphorylation of interferon regulatory factor 3 (IRF3), both of which constitutively associate with cytosolic chaperone Hsp90. It remains largely unknown how MAVS activates TBK1/IRF3. In this study, we identified translocases of outer membrane 70 (Tom70), a mitochondrial import receptor, to interact with MAVS upon RNA virus infection. Ectopic expression or knockdown of Tom70 could enhance or impair IRF3-mediated gene expression, respectively. Mechanistically, the clamp domain (R192) of Tom70 interacts with the C-terminal motif (EEVD) of Hsp90, thus recruiting TBK1/IRF3 to mitochondria. Disruption of this interaction or mislocation of Tom70 sharply impairs activation of TBK1 and IRF3. Furthermore, host antiviral responses are significantly boosted or crippled in the presence or absence of Tom70. Collectively, our study characterizes Tom70 as a critical adaptor linking MAVS to TBK1/IRF3, revealing that mitochondrion is evolutionarily integrated with innate immunity.