RESUMEN
RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Interferón Tipo I/genética , Proteínas de Transporte de Membrana/metabolismo , ARN no Traducido/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adulto , Anciano , Capa Leucocitaria de la Sangre/citología , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/sangre , Interferón Tipo I/inmunología , Macrófagos , Masculino , Persona de Mediana Edad , Fosforilación/genética , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , ARN no Traducido/sangre , ARN no Traducido/genética , RNA-Seq , Infecciones del Sistema Respiratorio/sangre , Infecciones del Sistema Respiratorio/microbiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Adulto JovenRESUMEN
Immortal hepatocyte cell lines are widely used to elucidate insulin-dependent signalling pathways and regulation of hepatic metabolism, although the often tumorigenic origin might not represent the metabolic state of healthy hepatocytes. We aimed to investigate if murine cell line AML12 and human cell line THLE-2, which are derived from healthy liver cells, are comparable to hepatoma cell line HepG2 for studying acute insulin signalling and expression of gluconeogenic enzymes and hepatokines. Insulin responsiveness of AML12 and THLE-2 cells was impaired when cells were cultured in the recommended growth medium, but comparable with HepG2 cells by using insulin-deficient medium. THLE-2 cells showed low abundance of insulin receptor, while protein levels in HepG2 and AML12 were comparable. AML12 and THLE-2 cells showed only low or non-detectable transcript levels of G6PC and PCK1 Expression of ANGPTL4 was regulated similarly in HepG2 and AML12 cells upon peroxisome proliferator-activated receptor δ activation but only HepG2 cells resemble the in vivo regulation of hepatic ANGPTL4 by cAMP. Composition of the culture medium and protein expression levels of key signalling proteins should be considered when AML12 and THLE-2 are used to study insulin signalling. With regard to gluconeogenesis and hepatokine expression, HepG2 cells appear to be closer to the in vivo situation despite the tumorigenic origin.