RESUMEN
The NF-κB essential modulator (NEMO) is a regulatory subunit of the IκB kinase (IKK) complex that phosphorylates the NF-κB inhibitors IκBs. NEMO mediates IKK activation by binding to polyubiquitin chains (polyUb). Here, we show that Lys63(K63)-linked or linear polyUb binding to NEMO robustly induced the formation of liquid-like droplets in which IKK was activated. This liquid phase separation of NEMO was driven by multivalent interactions between NEMO and polyUb. Both the NEMO ubiquitin-binding (NUB) domain and the zinc-finger (ZF) domain of NEMO mediated binding to polyUb and contributed to NEMO phase separation and IKK activation in cells. Moreover, NEMO mutations associated with human immunodeficiency impaired its phase separation. These results demonstrate that polyUb activates IKK and NF-κB signaling by promoting the phase separation of NEMO.
Asunto(s)
FN-kappa B , Poliubiquitina , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Poliubiquitina/genética , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection1-3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP)4,5, which leads to the expression of antimicrobial genes6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered8-11. These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively8,12. Although these proteins are required to prevent infection of some bacteriophages8, the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.
Asunto(s)
Bacterias , Proteínas Bacterianas , Bacteriófagos , Nucleotidiltransferasas , Ubiquitina , Animales , Bacterias/enzimología , Bacterias/inmunología , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/inmunología , Nucleótidos Cíclicos/biosíntesis , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Ubiquitina/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Virales/metabolismo , Interacciones Microbiota-HuespedRESUMEN
Circular RNAs (circRNAs) constitute a large class of RNA species formed by the back-splicing of co-linear exons, often within protein-coding transcripts. Despite much progress in the field, it remains elusive whether the majority of circRNAs are merely aberrant splicing by-products with unknown functions, or their production is spatially and temporally regulated to carry out specific biological functions. To date, the majority of circRNAs have been cataloged in resting cells. Here, we identify an LPS-inducible circRNA: mcircRasGEF1B, which is predominantly localized in cytoplasm, shows cell-type specific expression, and has a human homolog with similar properties, hcircRasGEF1B. We show that knockdown of the expression of mcircRasGEF1B reduces LPS-induced ICAM-1 expression. Additionally, we demonstrate that mcircRasGEF1B regulates the stability of mature ICAM-1 mRNAs. These findings expand the inventory of functionally characterized circRNAs with a novel RNA species that may play a critical role in fine-tuning immune responses and protecting cells against microbial infection.
Asunto(s)
Regulación de la Expresión Génica , Molécula 1 de Adhesión Intercelular/genética , ARN/genética , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/genética , Animales , Secuencia de Bases , Línea Celular , Evolución Molecular , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Especificidad de Órganos/genética , Transporte de Proteínas , ARN/química , Estabilidad del ARN , ARN Circular , Factores de Intercambio de Guanina Nucleótido ras/químicaRESUMEN
Transcriptional homeostasis relies on the balance between positive and negative regulation of gene transcription. Methylation of histone H3 lysine 9 (H3K9) is commonly correlated with gene repression. Here, we report that a euchromatic H3K9 methyltransferase, EHMT1, functions as a negative regulator in both the NF-κB- and type I interferon-mediated gene induction pathways. EHMT1 catalyzes H3K9 methylation at promoters of NF-κB target genes. Moreover, EHMT1 interacts with p50, and, surprisingly, p50 appears to repress the expression of type I interferon genes and genes activated by type I interferons by recruiting EHMT1 to catalyze H3K9 methylation at their promoter regions. Silencing the expression of EHMT1 by RNA interference enhances expression of a subset NF-κB-regulated genes, augments interferon production, and augments antiviral immunity.
Asunto(s)
Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Represoras/metabolismo , Animales , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/inmunología , Histonas/genética , Histonas/inmunología , Histonas/metabolismo , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Metilación , Ratones , Ratones Mutantes , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/inmunología , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Virosis/genética , Virosis/inmunología , Virosis/metabolismoRESUMEN
Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs. Key features This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study. Protocol for amplification of low levels of circRNA expression. Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.
RESUMEN
NF-kappaB is a key activator of inflammatory and immune responses with important pathological roles in cancer, heart disease, and autoimmune diseases. Transcriptional activity of NF-kappaB is regulated by different posttranslational modifications. Here, we report a novel mechanism of NF-kappaB regulation through lysine monomethylation by SET9 methyltransferase. Set9 specifically methylates p65 at lysine 37. Both TNFalpha and IL-1beta treatments induced methylation of p65. Methylated p65 is restricted to the nucleus and this modification regulates the promoter binding of p65. Moreover, Set9 mediated methylation of p65 is required for the expression of a subset of NF-kappaB target genes in response to TNFalpha stimulation.
Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Interleucina-1beta/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.
Asunto(s)
Herpesvirus Humano 4 , ARN Circular , Línea Celular , HumanosRESUMEN
Jumonji C (JmjC) domain-containing proteins have been shown to regulate cellular processes by hydroxylating or demethylating histone and non-histone targets. JMJD8 belongs to the JmjC domain-only family that was recently shown to be involved in angiogenesis and TNF-induced NF-κB signaling. Here, we employed bioinformatic analysis and immunofluorescence microscopy to examine the physiological properties of JMJD8. We demonstrated that JMJD8 localizes to the lumen of endoplasmic reticulum and that JMJD8 forms dimers or oligomers in vivo. Furthermore, we identified potential JMJD8-interacting proteins that are known to regulate protein complex assembly and protein folding. Taken together, this work demonstrates that JMJD8 is the first JmjC domain-containing protein found in the lumen of the endoplasmic reticulum that may function in protein complex assembly and protein folding.
Asunto(s)
Retículo Endoplásmico/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Biología Computacional , Células HEK293 , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/química , Oxidorreductasas N-Desmetilantes/química , Dominios Proteicos , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections.
Asunto(s)
Perfilación de la Expresión Génica , Lipopolisacáridos/metabolismo , ARN/metabolismo , Receptor Toll-Like 4/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/genética , Animales , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Células RAW 264.7 , ARN CircularRESUMEN
Influenza virus is estimated to cause 3-5 million severe complications and about 250-500 thousand deaths per year. Different kinds of anti-influenza virus drugs have been developed. However, the emergence of drug resistant strains has presented a big challenge for efficient antiviral therapy. Indole derivatives have been shown to exhibit both antiviral and anti-inflammatory activities. In this study, we adopted a cell-based system to screen for potential anti-IAV agents. Four indole derivatives (named 525A, 526A, 527A and 528A) were subjected to the antiviral screening, of which 526A was selected for further investigation. We reported that pre-treating cells with 526A protects cells from IAV infection. Furthermore, 526A inhibits IAV replication by inhibiting the expression of IAV genes. Interestingly, 526A suppresses the activation of IRF3 and STAT1 in host cells and thus represses the production of type I interferon response and cytokines in IAV-infected cells. Importantly, 526A also partially blocks the activation of RIG-I pathway. Taken together, these results suggest that 526A may be a potential anti-influenza A virus agent.
Asunto(s)
Antivirales/farmacología , Indoles/farmacología , Virus de la Influenza A/efectos de los fármacos , Malondialdehído/análogos & derivados , Animales , Humanos , Virus de la Influenza A/fisiología , Factor 3 Regulador del Interferón/metabolismo , Malondialdehído/farmacología , Factor de Transcripción STAT1/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
TNF-induced signaling mediates pleiotropic biological consequences including inflammation, immunity, cell proliferation and apoptosis. Misregulation of TNF signaling has been attributed as a major cause of chronic inflammatory diseases and cancer. Jumonji domain-containing protein 8 (JMJD8) belongs to the JmjC family. However, only part of the family members has been described as hydroxylase enzymes that function as histone demethylases. Here, we report that JMJD8 positively regulates TNF-induced NF-κB signaling. Silencing the expression of JMJD8 using RNA interference (RNAi) greatly suppresses TNF-induced expression of several NF-κB-dependent genes. Furthermore, knockdown of JMJD8 expression reduces RIP ubiquitination, IKK kinase activity, delays IκBα degradation and subsequently blocks nuclear translocation of p65. In addition, JMJD8 deficiency enhances TNF-induced apoptosis. Taken together, these findings indicate that JMJD8 functions as a positive regulator of TNF-induced NF-κB signaling.
RESUMEN
Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity in vivo and in vitro. NiPT5 impedes the signaling cascades that lead to the activation of NF-κB in response to different stimuli, such as LPS and TNFα. Using our cell-based screening system, we report that pretreating cells with NiPT5 protects cells from influenza A virus (IAV) and vesicular stomatitis virus (VSV) infection. Furthermore, NiPT5 inhibits replication of IAV by inhibiting transcription and translation of vRNAs of IAV. Additionally, NiPT5 reduces IAV-induced type I interferon response and cytokines production. Moreover, NiPT5 prevents activation of NF-κB, and IRF3 in response to IAV infection. These results demonstrate that NiPT5 is a potent antiviral agent that inhibits the early phase of IAV replication.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Virus de la Influenza A/efectos de los fármacos , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferones/metabolismo , FN-kappa B/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
In this study, we showed that suppression of the MEK-ERK transduction pathway by a selective inhibitor, 2'-amino-3'-methoxyflavone (PD98059), increased drug resistance of SiHa cells to cisplatin, but not to another common anticancer drug, doxorubicin. The downstream mechanism of this discrepant cellular response was investigated. Both cisplatin and doxorubicin activated nuclear ERK2 and nuclear transcription factor kappa B (NF kappa B) of SiHa cells. However, suppression of the MEK-ERK2 pathway by PD98059 resulted in a further enhancement of cisplatin-induced NF kappa B activation, while no further regulation of NF kappa B was noted in doxorubicin-treated cells. The activation of NF kappa B by cisplatin or doxorubicin was not due to the degradation of cytoplasmic I kappa B alpha, as demonstrated by western blotting. Transfection of a dominant negative I kappa B alpha resulted in a markedly diminished PD98059-induced cisplatin resistance in SiHa cells. Our results suggest that the MEK-ERK signaling pathway plays a role in the chemosensitivity of SiHa cells, and suppression of this pathway increases cisplatin resistance partly via an increase of NF kappa B activation. The mechanism responsible for the discrepant effect of PD98059 on NF kappa B activation and hence the chemosensitivity of SiHa cells towards cisplatin and doxorubicin remains to be investigated.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas I-kappa B , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/farmacología , Interacciones Farmacológicas , Femenino , Flavonoides/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , Transfección , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response. RESULTS: We report the application of EHMT1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNα2a in four CML cell lines, K562, KCL22, BV173 and KT1 cells. Chromatin immunoprecipitation assay shows that BIX01294 treatment enhances type I interferon response by reducing H3K9me2 at the promoters of interferon-stimulated genes. Additionally, BIX01294 treatment augments IFNα2a- and imatinib-mediated apoptosis in CML cell lines. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon responsiveness in CML cell lines. CONCLUSIONS: Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.
Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Interferón Tipo I/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Quinazolinas/farmacología , Apoptosis , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Expresión Génica/efectos de los fármacos , Células HeLa , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Piperazinas/farmacología , Pirimidinas/farmacologíaRESUMEN
BACKGROUND: The biological properties of thiosemicarbazone have been widely reported. The incorporation of some transition metals such as Fe, Ni and Cu to thiosemicarbazone complexes is known to enhance its biological effects. In this study, we incorporated nickel(II) ions into thiosemicarbazone with N4-substitution groups H3L (H; H3L1, CH3; H3L2, C6H5; H3L3 and C2H5; H3L4) and examined its potential anti-inflammatory activity. METHODOLOGY/PRINCIPAL FINDINGS: Four ligands (1-4) and their respective nickel-containing complexes (5-8) were synthesized and characterized. The compounds synthesized were tested for their effects on NF-κB nuclear translocation, pro-inflammatory cytokines secretion and NF-κB transactivation activity. The active compound was further evaluated on its ability to suppress carrageenan-induced acute inflammation in vivo. A potential binding target of the active compound was also predicted by molecular docking analysis. CONCLUSIONS/SIGNIFICANCE: Among all synthesized compounds tested, we found that complex [Ni(H2L1)(PPh3)]Cl (5) (complex 5), potently inhibited IκBα degradation and NF-κB p65 nuclear translocation in LPS-stimulated RAW264.7 cells as well as TNFα-stimulated HeLa S3 cells. In addition, complex 5 significantly down-regulated LPS- or TNFα-induced transcription of NF-κB target genes, including genes that encode the pro-inflammatory cytokines TNFα, IFNß and IL6. Luciferase reporter assays confirmed that complex 5 inhibited the transactivation activity of NF-κB. Furthermore, the anti-inflammatory effect of complex 5 was also supported by its suppressive effect on carrageenan-induced paw edema formation in wild type C57BL/6 mice. Interestingly, molecular docking study showed that complex 5 potentially interact with the active site of IKKß. Taken together, we suggest complex 5 as a novel NF-κB inhibitor with potent anti-inflammatory effects.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Complejos de Coordinación/farmacología , Inflamación/tratamiento farmacológico , Níquel/química , Tiosemicarbazonas/química , Activación Transcripcional/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Carragenina , Línea Celular , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Células HeLa , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Interferón beta/biosíntesis , Interferón beta/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Transporte de Proteínas , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
T cell responses in mammals must be tightly regulated to both provide effective immune protection and avoid inflammation-induced pathology. NF-κB activation is a key signaling event induced by T cell receptor (TCR) stimulation. Dysregulation of NF-κB is associated with T cell-mediated inflammatory diseases and malignancies, highlighting the importance of negative feedback control of TCR-induced NF-κB activity. In this study we show that in mice, T cells lacking miR-146a are hyperactive in both acute antigenic responses and chronic inflammatory autoimmune responses. TCR-driven NF-κB activation up-regulates the expression of miR-146a, which in turn down-regulates NF-κB activity, at least partly through repressing the NF-κB signaling transducers TRAF6 and IRAK1. Thus, our results identify miR-146a as an important new member of the negative feedback loop that controls TCR signaling to NF-κB. Our findings also add microRNA to the list of regulators that control the resolution of T cell responses.
Asunto(s)
MicroARNs/genética , Linfocitos T/fisiología , Animales , Antígenos/inmunología , Antígenos/farmacología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Regulación de la Expresión Génica , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , MicroARNs/metabolismo , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.
Asunto(s)
Proteínas Portadoras/metabolismo , Quinasa I-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poliubiquitina/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Línea Celular , Activación Enzimática , Humanos , Células Jurkat , Lisina/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/deficiencia , Proteínas de Complejo Poro Nuclear/genética , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Homología de Secuencia de AminoácidoRESUMEN
Viral infection triggers host innate immune responses through activation of the transcription factors NF-kappaB and IRF 3, which coordinately regulate the expression of type-I interferons such as interferon-beta (IFN-beta). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-kappaB and IRF 3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-kappaB and IRF 3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-beta through activation of NF-kappaB and IRF 3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF 3 and IkappaB and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inmunidad Innata , Proteínas de la Membrana/fisiología , Mitocondrias/inmunología , Proteínas Mitocondriales/fisiología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación Bacteriana de la Expresión Génica/genética , Silenciador del Gen , Células HeLa , Humanos , Factor 3 Regulador del Interferón , Factor 7 Regulador del Interferón , Interferón beta/inmunología , Interferones/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/inmunología , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/inmunología , ARN Bicatenario/inmunología , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Virus Sendai/genética , Virus Sendai/inmunología , Alineación de Secuencia , Replicación Viral/inmunologíaRESUMEN
The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.