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1.
J Biol Chem ; 299(10): 105213, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37660925

RESUMEN

The cyclic GMP-AMP synthase and stimulator of interferon (IFN) genes (cGAS-STING) pathway serves as a crucial component of innate immune defense and exerts immense antiviral activity by inducing the expression of type I IFNs. Currently, STING-activated production of type I IFNs has been thought to be mediated only by TANK-binding kinase 1 (TBK1). Here, we identified that porcine IKKε (pIKKε) is also directly involved in STING-induced type I IFN expression and antiviral response by using IKKε-/- porcine macrophages. Similar to pTBK1, pIKKε interacts directly with pSTING on the C-terminal tail. Furthermore, the TBK1-binding motif of pSTING C-terminal tail is essential for its interaction with pIKKε, and within the TBK1-binding motif, the leucine (L) 373 is also critical for the interaction. On the other hand, both kinase domain and scaffold dimerization domain of pIKKε participate in the interactions with pSTING. Consistently, the reconstitution of pIKKε and its mutants in IKKε-/- porcine macrophages corroborated that IKKε and its kinase domain and scaffold dimerization domain are all involved in the STING signaling and antiviral function. Thus, our findings deepen the understanding of porcine cGAS-STING pathway, which lays a foundation for effective antiviral therapeutics against porcine viral diseases.

2.
J Biol Chem ; 299(7): 104925, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37328105

RESUMEN

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1 cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1 cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis.


Asunto(s)
Infecciones por VIH , Interferón Tipo I , Proteína 1 que Contiene Dominios SAM y HD , Humanos , Células HEK293 , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Infecciones por VIH/metabolismo , Transducción de Señal
3.
EMBO J ; 39(17): e104202, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32696476

RESUMEN

IL-17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL-17 receptor (IL-17R) remained elusive. We developed a novel mass spectrometry-based approach to identify components of the IL-17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL-17R, we established that IL-17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL-17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL-17R complex, documenting that NEMO has an unprecedented negative function in IL-17 signaling, distinct from its role in NF-κB activation. Our study provides a comprehensive view of the molecular events of the IL-17 signal transduction and its regulation.


Asunto(s)
Retroalimentación Fisiológica , Receptores de Interleucina-17/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Interleucina-17/genética
4.
J Gen Virol ; 105(8)2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39167082

RESUMEN

Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes a highly common but mild infection characterized by distinctive and persistent papular skin lesions. These lesions can persist for long periods without an effective clearance response from the host. MCV, like all poxviruses, encodes multiple known immunosuppressive proteins which target innate immune signalling pathways involved in viral nucleic acid sensing, interferon production and inflammation which should trigger antiviral immunity leading to clearance. Two major families of transcription factors responsible for driving the immune response to viruses are the NF-κB and the interferon regulatory factor (IRF) families. While NF-κB broadly drives pro-inflammatory gene expression and IRFs chiefly drive interferon induction, both collaborate in transactivating many of the same genes in a concerted immune response to viral infection. Here, we report that the MCV protein MC089 specifically inhibits IRF activation from both DNA- and RNA-sensing pathways, making it the first characterized MCV inhibitor to selectively target IRF activation to date. MC089 interacts with proteins required for IRF activation, namely IKKε, TBKBP1 and NAP1. Additionally, MC089 targets RNA sensing by associating with the RNA-sensing adaptor protein mitochondrial antiviral-signalling protein on mitochondria. MC089 displays specificity in its inhibition of IRF3 activation by suppressing immunostimulatory nucleic acid-induced serine 396 phosphorylation without affecting the phosphorylation of serine 386. The selective interaction of MC089 with IRF-regulatory proteins and site-specific inhibition of IRF3 phosphorylation may offer a tool to provide novel insights into the biology of IRF3 regulation.


Asunto(s)
Factor 3 Regulador del Interferón , Virus del Molusco Contagioso , Proteínas Virales , Humanos , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Virus del Molusco Contagioso/inmunología , Virus del Molusco Contagioso/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/inmunología , Transducción de Señal , Inmunidad Innata , Células HEK293 , Interacciones Huésped-Patógeno/inmunología
5.
J Virol ; 97(12): e0150123, 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-37982618

RESUMEN

IMPORTANCE: The type-I interferon (IFN-I) signaling pathway is the first line of antiviral innate immunity. It must be precisely regulated against virus-induced damage. The tightly regulated mechanisms of action of host genes in the antiviral innate immune signaling pathway are still worth studying. Here, we report a novel role of DLG1 in positively regulating the IκB kinase epsilon (IKKε)-mediated IFN-I signaling response against negative-stranded RNA virus replication, whereas the RNA virus inhibits the expression of DLG1 for immune escape. Importantly, the E3 ligase March2 interacts with and promotes K27-linked polyubiquitination of IKKε, and p62 is a cargo receptor that recognizes ubiquitinated IKKε for eventual autophagic degradation. Together, the current findings elucidate the role of DLG1 in the antiviral IFN-I signaling pathway and viral infection repression.


Asunto(s)
Autofagia , Homólogo 1 de la Proteína Discs Large , Quinasa I-kappa B , Inmunidad Innata , Virus ARN de Sentido Negativo , Proteína Sequestosoma-1 , Virosis , Humanos , Homólogo 1 de la Proteína Discs Large/metabolismo , Quinasa I-kappa B/metabolismo , Inmunidad Innata/inmunología , Virus ARN de Sentido Negativo/crecimiento & desarrollo , Virus ARN de Sentido Negativo/inmunología , Poliubiquitina/metabolismo , Proteína Sequestosoma-1/antagonistas & inhibidores , Transducción de Señal , Virosis/inmunología , Animales , Línea Celular
6.
Inflamm Res ; 73(6): 897-913, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38625657

RESUMEN

OBJECTIVES AND DESIGN: As an interferon-inducible protein, Viperin has broad-spectrum antiviral effects and regulation of host immune responses. We aim to investigate how Viperin regulates interferon-γ (IFN-γ) production in macrophages to control Mycobacterium tuberculosis (Mtb) infection. METHODS: We use Viperin deficient bone-marrow-derived macrophage (BMDM) to investigate the effects and machines of Viperin on Mtb infection. RESULTS: Viperin inhibited IFN-γ production in macrophages and in the lung of mice to promote Mtb survival. Further insight into the mechanisms of Viperin-mediated regulation of IFN-γ production revealed the role of TANK-binding kinase 1 (TBK1), the TAK1-dependent inhibition of NF-kappa B kinase-epsilon (IKKε), and interferon regulatory factor 3 (IRF3). Inhibition of the TBK1-IKKε-IRF3 axis restored IFN-γ production reduced by Viperin knockout in BMDM and suppressed intracellular Mtb survival. Moreover, Viperin deficiency activated the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which promoted IFN-γ production and inhibited Mtb infection in BMDM. Additionally, a combination of the anti-TB drug INH treatment in the absence of Viperin resulted in further IFN-γ production and anti-TB effect. CONCLUSIONS: This study highlights the involvement of TBK1-IKKε-IRF3 axis and JAK-STAT signaling pathways in Viperin-suppressed IFN-γ production in Mtb infected macrophages, and identifies a novel mechanism of Viperin on negatively regulating host immune response to Mtb infection.


Asunto(s)
Factor 3 Regulador del Interferón , Interferón gamma , Macrófagos , Ratones Endogámicos C57BL , Mycobacterium tuberculosis , Proteínas Serina-Treonina Quinasas , Proteínas , Transducción de Señal , Animales , Interferón gamma/metabolismo , Interferón gamma/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Mycobacterium tuberculosis/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Ratones , Proteínas/genética , Proteínas/metabolismo , Quinasa I-kappa B/metabolismo , Quinasas Janus/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Ratones Noqueados , Tuberculosis/inmunología , Pulmón/inmunología , Pulmón/microbiología , Proteína Viperina
7.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-34518217

RESUMEN

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.


Asunto(s)
Quinasa I-kappa B/genética , Inflamasomas/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Animales , Línea Celular , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/genética
8.
J Virol ; 96(22): e0112522, 2022 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-36326274

RESUMEN

Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The phosphoprotein (P) of lyssaviruses (lyssavirus-P) plays multiple roles in virus replication and immune evasion. Lyssavirus-P has been identified as the major type I interferon (IFN-I) antagonist, while the precise site and precise molecular mechanism remain unclear. Herein, we found that substitution of site 179 of lyssavirus-P from serine (Ser) to proline (Pro) impairs its antagonism function of IFN-I by sequence alignment and site mutations. Subsequent studies demonstrated that lyssavirus-P containing S179 specifically interacted with I-kappa B kinase ε (IKKε). Specifically, lyssavirus-P containing S179 interacted simultaneously with the kinase domain (KD) and scaffold dimerization domain (SDD) of IKKε, competing with TNF receptor-associated factor 3 (TRAF3) and IFN regulatory factor 3 (IRF3) for binding with IKKε, leading to the inhibition of IFN production. Furthermore, S179 was involved in the viral pathogenicity of the typical lyssavirus rabies virus in a mouse model. Interestingly, we found that S179 is conserved among most lyssavirus-P and functional for IFN antagonism. Collectively, we identified S179 of lyssavirus-P is essential for IFN-I inhibition, which provides deep insight into the immune evasion strategies of lyssaviruses. IMPORTANCE Interferon (IFN) and the IFN-induced cellular antiviral response constitute the first line of defense against viral invasion. Evading host innate immunity, especially IFN signaling, is the key step required for lyssaviruses to establish infection. In this study, S179 of lyssavirus phosphoprotein (lyssavirus-P) was identified as the key site for antagonizing IFN-I production. Mechanistically, lyssavirus-P containing S179 specifically targets the key kinase IKKε and disrupts its interaction with TRAF3 and IRF3. S179P mutation in the P protein of the typical lyssavirus rabies virus (RABV) attenuated its pathogenicity in a mouse model. Our findings provide deep insight into the immune evasion strategies of lyssaviruses, which is helpful for the development of effective antiviral therapeutics.


Asunto(s)
Interferón Tipo I , Lyssavirus , Virus de la Rabia , Animales , Ratones , Humanos , Lyssavirus/genética , Quinasa I-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Interferón Tipo I/metabolismo , Antivirales
9.
Fish Shellfish Immunol ; 142: 109095, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37730077

RESUMEN

IKKε plays an important role in the activation of IRF3/IRF7 and the production of interferon (IFN), however, its regulation remains obscure in human. E3 ligase TRIM25 has been reported to manipulate the K63-linked ubiquitination of RIG-I, leading to the activation of RIG-I/IFN signaling. To elucidate the role of TRIM25 in teleost, a TRIM25 homolog (bcTRIM25) was cloned and characterized from black carp (Mylopharyngodon piceus). bcTRIM25 contains 653 amino acids, possessing conservative RING, B-box and SPRY domain, which is highly expressed in muscle, spleen and skin. bcTRIM25 knock-down enhanced the antiviral ability of host cells. bcTRIM25 over-expression alone in EPC cells attenuated bcIFNa promoter transcription in the reporter assays and impeded PKR and MX1 expression in qRT-PCR. Interestingly, co-IP assays indicated that bcTRIM25 interacted with bcIKKε and the induced bcIFNa promoter transcription by bcIKKε was notably hindered by bcTRIM25. Furthermore, bcIKKε-induced expression of interferon stimulated genes (ISGs) and antiviral activity were dampened by bcTRIM25. Further exploration showed that bcTRIM25 visibly enhanced the ubiquitination of bcIKKε but significantly attenuated the phosphorylation of bcIKKε. Thus, our data demonstrate for the first time in vertebrate that TRIM25 negatively regulates IKKε through enhancing its ubiquitination, which sheds a light on the regulation of IKKε/IFN signaling.


Asunto(s)
Carpas , Quinasa I-kappa B , Animales , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/química , Carpas/genética , Carpas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Interferones/genética , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Antivirales , Inmunidad Innata/genética , Factores de Transcripción
10.
Biochem Biophys Res Commun ; 589: 267-274, 2022 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-34933200

RESUMEN

The deprivation of myocardial nutrition causes cardiomyocyte death and disturbance of energy metabolism. IKKε plays an important regulatory role in many biological events such as inflammation, redox reaction, cell death, etc. However, the more in-depth mechanism by which IKKε contributes to cardiomyocytes death in nutrition deprivation remains poorly understood. IKKε expression was knocked down by siRNA in H9c2 cells, and cells were cultured under starvation conditions to simulate ischemic conditions. Starvation triggered greater NLRP3 activation, accompanied by more IL-1ß, IL-18 and caspase-1 release in the siIKKε H9c2 cells compared with the control H9c2 cells. Western blot and immunofluorescence showed that the IKKε konckdown promoted NLRP3 expressions and ROS release under starvation conditions. Furthermore, electron micrography and JC-1 analysis revealed that IKKε konckdown resulted in aggravated mitochondrial damage and more mitochondrial ROS (mtROS) released in vitro. Notably, Western blot analysis showed that IKKε deficiency activated the TBK1 and IRF3 signaling pathways to promote pyroptosis in vitro. Collectively, our results indicate that IKKε protects against cardiomyocyte injury by reducing mitochondrial damage and NLRP3 expression following nutrition deprivation via regulation of the TBK1/IRF3 signaling pathway. This study further revealed the mechanism of IKKε in inflammation and myocardial nutrition deprivation.


Asunto(s)
Citoprotección , Quinasa I-kappa B/metabolismo , Inflamasomas/metabolismo , Mitocondrias/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Quinasa I-kappa B/deficiencia , Factor 3 Regulador del Interferón/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas
11.
J Virol ; 95(19): e0092321, 2021 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-34260286

RESUMEN

Peroxiredoxin 1 (PRDX1) is a cellular antioxidant enzyme that is crucial for diverse fundamental biological processes, such as autophagy, inflammation, and carcinogenesis. However, molecular mechanisms underpinning its diverse roles are not well understood. Here, we report that PRDX1 positively regulates interferon (IFN) induction and that pseudorabies virus (PRV) targets PRDX1 to evade IFN induction. PRV UL13 encodes a serine/threonine kinase important for PRV infection, although its biological function remains obscure. We identified PRDX1 as a UL13-interacting protein. Virological and biochemical assays demonstrate that PRDX1 promotes IFN induction by interacting with TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε). Conversely, UL13 accelerates PRDX1 degradation via the ubiquitin-proteosome pathway in a kinase-dependent manner. In doing so, PRV inhibits IFN induction during productive infection, which requires PRDX1 expression. This study uncovers an essential role of PRDX1 in the innate immune response and reveals a new viral immune evasion strategy to counteract cellular defenses. IMPORTANCE PRV interacts with numerous cellular proteins during productive infection. Here, we demonstrated the interaction of viral protein UL13 with the antioxidant enzyme PRDX1, which functions in multiple signal transduction pathways. We found that PRDX1 participates in the type I IFN pathway by interacting with TBK1 and IKKε, thereby negatively regulating PRV propagation. However, UL13 ubiquitinates PRDX1, which routes PRDX1 into proteasomes for degradation and effectively reduces its expression. These results illuminate the fundamental role that PRDX1 plays in the IFN pathway, and they identify a potential target for the control of PRV infection.


Asunto(s)
Herpesvirus Suido 1/fisiología , Quinasa I-kappa B/metabolismo , Inmunidad Innata , Peroxirredoxinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Células HEK293 , Herpesvirus Suido 1/inmunología , Humanos , Evasión Inmune , Interferón Tipo I/biosíntesis , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitinación , Proteínas Virales/genética , Replicación Viral
12.
EMBO Rep ; 21(9): e48260, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32783398

RESUMEN

IκB kinase ε (IKKε) is a key molecule at the crossroads of inflammation and cancer. Known to regulate cytokine secretion via NFκB and IRF3, the kinase is also a breast cancer oncogene, overexpressed in a variety of tumours. However, to what extent IKKε remodels cellular metabolism is currently unknown. Here, we used metabolic tracer analysis to show that IKKε orchestrates a complex metabolic reprogramming that affects mitochondrial metabolism and consequently serine biosynthesis independently of its canonical signalling role. We found that IKKε upregulates the serine biosynthesis pathway (SBP) indirectly, by limiting glucose-derived pyruvate utilisation in the TCA cycle, inhibiting oxidative phosphorylation. Inhibition of mitochondrial function induces activating transcription factor 4 (ATF4), which in turn drives upregulation of the expression of SBP genes. Importantly, pharmacological reversal of the IKKε-induced metabolic phenotype reduces proliferation of breast cancer cells. Finally, we show that in a highly proliferative set of ER negative, basal breast tumours, IKKε and PSAT1 are both overexpressed, corroborating the link between IKKε and the SBP in the clinical context.


Asunto(s)
Neoplasias de la Mama , Quinasa I-kappa B , Mitocondrias , Serina/biosíntesis , Neoplasias de la Mama/genética , Femenino , Humanos , Quinasa I-kappa B/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Oncogenes/genética
13.
Drug Dev Res ; 83(1): 194-207, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34350600

RESUMEN

Nuclear factor kappa B cells (NF-κB) activation causes induction of the noncanonical IκB kinases (I-kappa-B kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) in liver and fat after high fat diet which followed activating of cascade of counter-inflammation that conserves energy storage. Chrysin (5,7-dihydroxyflavone), a natural flavonoid, present in many plants, honey and propolis, used conventionally to treat numerous ailments. The present study was aimed to identify the protective role of chrysin on the glucose lowering and insulin sensitivity in diet induced obese (DIO) mice by regulating IKKε/TBK1. Chrysin administered therapeutically (60, 100, 200 mg/kg body weight) and preventive mode (200 mg/kg body weight) for 4 and 10 weeks respectively to DIO mice. At last fasting blood glucose, oral glucose tolerance test, serum lipid profile, as well as the expression level of IKKε/TBK1 and triglyceride in the liver tissue were assessed. DIO mice showed impaired glucose tolerance, reduced weight gain, elevated hepatic IKKε/TBK1 expression, fatty acid infiltration triglyceride and increased in plasma insulin and glucose. Chrysin in both therapeutic and preventive mode normalized the altered levels of the same. Overall chrysin improves glycemic control and insulin sensitivity through regulating expression of IKKε/TBK1 in liver of DIO mice.


Asunto(s)
Quinasa I-kappa B , Resistencia a la Insulina , Animales , Dieta Alta en Grasa/efectos adversos , Flavonoides/farmacología , Quinasa I-kappa B/metabolismo , Hígado/metabolismo , Ratones , Ratones Obesos , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas
14.
Cytotherapy ; 23(9): 787-792, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34119434

RESUMEN

BACKGROUND AIMS: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.


Asunto(s)
Quinasa I-kappa B , Estomatitis Vesicular , Animales , Vectores Genéticos/genética , Humanos , Quinasa I-kappa B/genética , Células Asesinas Naturales , Lentivirus/genética , Leucocitos Mononucleares , Proteínas Serina-Treonina Quinasas/genética , Transducción Genética
15.
Fish Shellfish Immunol ; 118: 160-168, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34500054

RESUMEN

Interferon regulatory factor 3 (IRF3) is activated by IκB kinase ε (IKKε) and Tank-binding kinase 1 (TBK1), which plays a crucial role in the interferon signaling in vertebrates. However, the regulation of teleost IRF3 by IKKε remains largely unknown. In this study, the IRF3 homologue (bcIRF3) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcIRF3 was detected to increase in host cells in response to different stimuli. bcIRF3 distributed predominantly in the cytosolic area; however, translocated into nuclei after virus infection. bcIRF3 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF3 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). Moreover, knockdown of bcIRF3 reduced the antiviral ability of the host cells, and the transcription of antiviral-related cytokines was obviously lower in bcIRF3-deficient host cells than that of control cells. The data of reporter assay and plaque assay demonstrated that bcIKKε obviously enhanced bcIRF3-mediated IFN production and antiviral activity. Immunofluorescent staining and co-immunoprecipitation assay revealed that bcIKKε interacted with bcIRF3. It was interesting that the nuclear translocation of bcIRF3 and bcIKKε was enhanced by each other when these two molecules were co-expressed in the cells, however, the protein levels of bcIRF3 and bcIKKε were decreased mutually. Thus, our data support the conclusion that bcIKKε interacts with bcIRF3 and enhances bcIRF3-mediated antiviral signaling during host innate immune activation.


Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Reoviridae , Infecciones por Rhabdoviridae , Secuencia de Aminoácidos , Animales , Antivirales , Carpas/genética , Carpas/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Quinasa I-kappa B/metabolismo , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Rhabdoviridae/veterinaria
16.
J Cell Mol Med ; 24(19): 11573-11582, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32858764

RESUMEN

The IKK-related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll-like receptors to regulate NF-κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B-cell lymphoma (DLBCL). DLBCL cell lines and patient-derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non-germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL-10 production from Ly10 and repressed NF-κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF-κB signalling in lymphoma.


Asunto(s)
Quinasa I-kappa B/metabolismo , Interleucina-10/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Quimiocinas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción ReIA/metabolismo
17.
Biochem Biophys Res Commun ; 532(3): 362-369, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-32883523

RESUMEN

Non-alcoholic fatty liver disease (NAFLD) was associated with increased level of lipopolysaccharides (LPS) which mechanism remained unclear on intervention between LPS and NAFLD. The aim was to explore the IKKε/NF-κB role and its intervention of LPS and high-fat diet (HFD) induced NAFLD. Male C57BL/6 mice were fed on high-fat diet (HFD) combined with or without simultaneously subcutaneous injection of LPS for 18 weeks. Body weight , blood biochemistry parameters, inflammatory mediator and liver lipid deposition were measured to evaluate LPS effect on NAFLD. Furthermore, IKKε selective inhibitor amlexanox (AM) was administrated by gavage to HFD + LPS induced mice. The indicators about metabolism and inflammation were examined and qRT-PCR, immunoblotting assay as well as immunohistochemistry were performed to assess IKKε/NF-κB activation and downstream gene expression. This study found that low-dose LPS + HFD aggravated more significant steatosis than simple HFD or high-dose LPS + HFD. Low-dose LPS exacerbated more prominent inflammation profile including increased IKKε and NF-κB expression in liver than HFD. Inhibiting IKKε/NF-κB signaling with amlexanox significantly prevented HFD + LPS induced metabolic disorders and hepatic steatosis. LPS-upregulated gene expression involved in glucolipid metabolism could be downregulated by amlexanox. Thus, the present study confirmed long-term combinational administration of subcutaneous low-dose LPS injection and HFD induced NAFLD model which had more significant phenotype in mice than simple HFD or high-dose LPS-induction. Targeting on IKKε/NF-κB signaling with its inhibitor amlexanox alleviated steatohepatitis, suggesting that IKKε/NF-κB signaling was responsible for effect of LPS and HFD on NAFLD.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Quinasa I-kappa B/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/efectos adversos , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Aminopiridinas/farmacología , Animales , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Quinasa I-kappa B/antagonistas & inhibidores , Inyecciones Subcutáneas , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos
18.
J Virol ; 93(21)2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31434731

RESUMEN

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKß, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKß, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication.IMPORTANCE Excessive innate immune responses can be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKKα, IKKß, and IKKε. IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKß, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.


Asunto(s)
Quinasa I-kappa B/metabolismo , Inmunidad Innata/inmunología , Interferones/farmacología , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Antivirales/farmacología , Retroalimentación Fisiológica , Humanos , Quinasa I-kappa B/genética , Inmunidad Innata/efectos de los fármacos , Gripe Humana/inmunología , Gripe Humana/patología , Gripe Humana/virología , FN-kappa B , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/virología , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/inmunología , Fosforilación , Homología de Secuencia , Transducción de Señal , Proteínas de Unión a Tacrolimus/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética
19.
Int J Mol Sci ; 21(13)2020 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-32630674

RESUMEN

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this "difficult-to-treat" cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.


Asunto(s)
Aminopiridinas/farmacología , Melanoma/metabolismo , Aminopiridinas/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/metabolismo , Melanoma/tratamiento farmacológico , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
20.
Development ; 143(20): 3806-3816, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27578797

RESUMEN

Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis.


Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa C/metabolismo , Actinas/genética , Animales , Proteínas Portadoras/genética , Drosophila , Proteínas de Drosophila/genética , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteínas de Microfilamentos/genética , Fosforilación , Proteína Quinasa C/genética , Transducción de Señal
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