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1.
JCI Insight ; 2024 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-39325541

RESUMO

Grover disease is an acquired epidermal blistering disorder in which keratinocytes lose intercellular connections. While its pathologic features are well-defined, its etiology remains unclear and it lacks any FDA-approved therapy. Interestingly, Grover disease was a common adverse event in clinical trials for cancer using B-RAF inhibitors, but it remained unknown how B-RAF blockade compromised skin integrity. Here we identified ERK hyperactivation as a key driver of Grover disease pathology. We leveraged a fluorescent biosensor to confirm that B-RAF inhibitors, dabrafenib and vemurafenib, paradoxically activated ERK in human keratinocytes and organotypic epidermis, disrupting cell-cell junctions and weakening epithelial integrity. Consistent with clinical data showing that concomitant MEK blockade prevents Grover disease in patients receiving B-RAF inhibitors, we found that MEK inhibition suppressed ERK and rescued cohesion of B-RAF-inhibited keratinocytes. Validating these results, we demonstrated ERK hyperactivation in patient biopsies from vemurafenib-induced Grover disease, but also from spontaneous Grover disease, revealing a common etiology for both. Finally, in line with our recent identification of ERK hyperactivation in Darier disease, a genetic disorder with identical pathology to Grover disease, our studies uncovered that the pathogenic mechanisms of these two diseases converge on ERK signaling and support MEK inhibition as a therapeutic strategy. .

2.
bioRxiv ; 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38746263

RESUMO

Grover disease is an acquired dermatologic disorder characterized by pruritic vesicular and eroded skin lesions. While its pathologic features are well-defined, including impaired cohesion of epidermal keratinocytes, the etiology of Grover disease remains unclear and it lacks any FDA-approved therapy. Interestingly, drug-induced Grover disease occurs in patients treated with B-RAF inhibitors that can paradoxically activate C-RAF and the downstream kinase MEK. We recently identified hyperactivation of MEK and ERK as key drivers of Darier disease, which is histologically identical to Grover disease, supporting our hypothesis that they share a pathogenic mechanism. To model drug-induced Grover disease, we treated human keratinocytes with clinically utilized B-RAF inhibitors dabrafenib or vemurafenib and leveraged a fluorescent biosensor to confirm they activated ERK, which disrupted intercellular junctions and compromised keratinocyte sheet integrity. Consistent with clinical data showing concomitant MEK blockade prevents Grover disease in patients receiving B-RAF inhibitors, we found that MEK inhibition suppressed excess ERK activity to rescue cohesion of B-RAF-inhibited keratinocytes. Validating these results, we demonstrated ERK hyperactivation in skin biopsies of vemurafenib-induced Grover disease, but also in spontaneous Grover disease. In sum, our data define a pathogenic role for ERK hyperactivation in Grover disease and support MEK inhibition as a therapeutic strategy.

3.
Sci Signal ; 17(827): eade3643, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38470955

RESUMO

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-ß and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.


Assuntos
Complexo de Golgi , Fosfolipídeos , Fosfolipídeos/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
4.
JCI Insight ; 8(18)2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37561594

RESUMO

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than 2 decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2-knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomics analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAPK signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings, with lesions showing keratin deficiency, cadherin mislocalization, and ERK hyperphosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multiomic analysis with human organotypic epidermis as a preclinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.


Assuntos
Doença de Darier , Camundongos , Animais , Humanos , Doença de Darier/tratamento farmacológico , Doença de Darier/genética , Doença de Darier/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Retículo Endoplasmático/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
5.
JCI Insight ; 8(12)2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37166992

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a DNA sensor and responsible for inducing an antitumor immune response. Recent studies reveal that cGAS is frequently inhibited in cancer, and therapeutic targets to promote antitumor cGAS function remain elusive. SRC is a proto-oncogene tyrosine kinase and is expressed at elevated levels in numerous cancers. Here, we demonstrate that SRC expression in primary and metastatic bladder cancer negatively correlates with innate immune gene expression and immune cell infiltration. We determine that SRC restricts cGAS signaling in human cell lines through SRC small molecule inhibitors, depletion, and overexpression. cGAS and SRC interact in cells and in vitro, while SRC directly inhibits cGAS enzymatic activity and DNA binding in a kinase-dependent manner. SRC phosphorylates cGAS, and inhibition of cGAS Y248 phosphorylation partially reduces SRC inhibition. Collectively, our study demonstrates that cGAS antitumor signaling is hindered by the proto-oncogene SRC and describes how cancer-associated proteins can regulate the innate immune system.


Assuntos
Neoplasias , Nucleotidiltransferases , Humanos , Nucleotidiltransferases/metabolismo , Imunidade Inata , Neoplasias/genética , DNA/metabolismo , Proto-Oncogenes
6.
bioRxiv ; 2023 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-36945477

RESUMO

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2 knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

7.
J Invest Dermatol ; 143(3): 353-361.e4, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36822769

RESUMO

Barrier tissues such as the epidermis employ complex signal transduction systems to execute morphogenetic programs and to rapidly respond to environmental cues to promote homeostasis. Recent advances in live-imaging techniques and tools allow precise spatial and temporal monitoring and manipulation of intracellular signaling cascades. Leveraging the chemistry of naturally occurring light-sensitive proteins, genetically encoded fluorescent biosensors have emerged as robust tools for visualizing dynamic signaling events. In contrast, optogenetic protein constructs permit laser-mediated control of signal receptors and effectors within live cells, organoids, and even model organisms. In this paper, we review the basic principles underlying novel biosensors and optogenetic tools and highlight how recent studies in cutaneous biology have leveraged these imaging strategies to illuminate the spatiotemporal signals regulating epidermal development, barrier formation, and tissue homeostasis.


Assuntos
Optogenética , Proteínas , Optogenética/métodos , Transdução de Sinais
8.
Elife ; 102021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34342578

RESUMO

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , Animais , COVID-19/imunologia , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Humanos , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/isolamento & purificação
9.
J Bacteriol ; 203(19): e0008021, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34309402

RESUMO

3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. IMPORTANCE c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.


Assuntos
Técnicas Biossensoriais , Fosfatos de Dinucleosídeos/química , Transferência Ressonante de Energia de Fluorescência , Nucleotídeos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica
10.
Elife ; 102021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33955352

RESUMO

Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Although studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here, we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS-dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise sensitive organism in vitro and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.


Assuntos
Aldeídos/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Animais , Bacillus subtilis/genética , Linhagem Celular , Feminino , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose
11.
Chembiochem ; 22(6): 1030-1041, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33142009

RESUMO

Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Bactérias/metabolismo , Fosfatos de Dinucleosídeos/análise , Ensaios Enzimáticos/métodos , Monofosfato de Adenosina/metabolismo , Bactérias/metabolismo , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Ensaios de Triagem em Larga Escala , Hidrólise , Luciferases/metabolismo , Medições Luminescentes/métodos , Mycobacterium tuberculosis/enzimologia
12.
Nat Commun ; 11(1): 3533, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669552

RESUMO

Cyclic dinucleotides (CDNs) are second messengers conserved across all three domains of life. Within eukaryotes they mediate protective roles in innate immunity against malignant, viral, and bacterial disease, and exert pathological effects in autoimmune disorders. Despite their ubiquitous role in diverse biological contexts, CDN detection methods are limited. Here, using structure guided design of the murine STING CDN binding domain, we engineer a Förster resonance energy transfer (FRET) based biosensor deemed BioSTING. Recombinant BioSTING affords real-time detection of CDN synthase activity and inhibition. Expression of BioSTING in live human cells allows quantification of localized bacterial and eukaryotic CDN levels in single cells with low nanomolar sensitivity. These findings establish BioSTING as a powerful kinetic in vitro platform amenable to high throughput screens and as a broadly applicable cellular tool to interrogate the temporal and spatial dynamics of CDN signaling in a variety of infectious, malignant, and autoimmune contexts.


Assuntos
Técnicas Biossensoriais , Proteínas de Membrana/química , Nucleotídeos Cíclicos/análise , Transdução de Sinais , Animais , Bacillus subtilis/química , GMP Cíclico/metabolismo , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunidade Inata , Ligantes , Camundongos , Mutagênese , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes
13.
Nature ; 579(7800): E12, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32144410

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

14.
Curr Opin Cell Biol ; 63: 49-56, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31958669

RESUMO

Cyclic dinucleotides (CDNs) have emerged as ubiquitous signaling molecules in all domains of life. In eukaryotes, CDN signaling systems are evolutionarily ancient and have developed to sense and respond to pathogen infection. On the other hand, dysregulation of these pathways has been implicated in the pathogenesis of autoimmune diseases. Thus, CDNs have garnered major interest over recent years for their ability to elicit potent immune responses in the eukaryotic host. Similarly, ancestral CDN-based signaling systems also appear to confer immunological protection against infection in prokaryotes. Therefore, a better understanding of the host processes regulated by CDNs will be of tremendous value in many areas of research. Here, we aim to review the latest discoveries and recent trends in CDN research with a particular focus on the molecular mechanisms by which these small molecules mediate innate immunity.


Assuntos
Imunidade Inata/imunologia , Nucleotídeos Cíclicos/metabolismo , Animais , Humanos
15.
Nature ; 573(7774): 434-438, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31511694

RESUMO

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway3. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)4-7. This cyclic dinucleotide (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells9 and other CDNs, including those produced by bacteria10-12 and synthetic CDNs used in cancer immunotherapy13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer13, host responsiveness to CDN-producing pathogenic microorganisms11 and-potentially-for some inflammatory diseases.


Assuntos
DNA/metabolismo , Nucleotídeos Cíclicos/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismo , Animais , Citosol , DNA/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Nucleotídeos Cíclicos/imunologia , Nucleotidiltransferases/metabolismo , Proteína Carregadora de Folato Reduzido/imunologia
16.
Nat Commun ; 9(1): 2770, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018345

RESUMO

Detection of viral genomes by the innate immune system elicits an antiviral gene program mediated by type I interferons (IFNs). While viral RNA and DNA species induce IFN via separate pathways, the mechanisms by which these pathways are differentially modulated are unknown. Here we show that the positive regulator of IFN in the RNA pathway, TRAF3, has an inhibitory function in the DNA pathway. Loss of TRAF3 coincides with increased expression of the alternative NF-κB-inducing molecule, NIK, which interacts with the DNA pathway adaptor, STING, to enhance IFN induction. Cells lacking NIK display defective IFN activation in the DNA pathway due to impaired STING signaling, and NIK-deficient mice are more susceptible to DNA virus infection. Mechanistically, NIK operates independently from alternative NF-κB signaling components and instead requires autophosphorylation and oligomerization to activate STING. Thus a previously undescribed pathway for NIK exists in activating IFN in the DNA pathway.


Assuntos
DNA Viral/genética , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Proteínas Serina-Treonina Quinases/genética , RNA Viral/genética , Fator 3 Associado a Receptor de TNF/genética , Vírus da Estomatite Vesicular Indiana/genética , Células A549 , Animais , DNA Viral/imunologia , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Células HEK293 , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Viral/imunologia , Transdução de Sinais , Células THP-1 , Fator 3 Associado a Receptor de TNF/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Quinase Induzida por NF-kappaB
17.
Methods Mol Biol ; 1280: 247-68, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25736753

RESUMO

Nuclear factor-κB (NF-κB) signaling is classified into the canonical and noncanonical pathways. We describe in this chapter the methods used to study the noncanonical pathway, including derivation of primary cells, pathway stimulation, and immunoblotting.


Assuntos
NF-kappa B/metabolismo , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Linfócitos B/metabolismo , Western Blotting , Técnicas de Cultura de Células , Ativação Enzimática , Fibroblastos/metabolismo , Separação Imunomagnética/métodos , Macrófagos/metabolismo , Camundongos , NF-kappa B/química , Cultura Primária de Células , Ligação Proteica , Transporte Proteico
18.
Nat Immunol ; 13(12): 1155-61, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23142775

RESUMO

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.


Assuntos
GMP Cíclico/análogos & derivados , RNA Helicases DEAD-box/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Interferon Tipo I/imunologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Linhagem Celular , GMP Cíclico/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores de Reconhecimento de Padrão/genética , Sistemas do Segundo Mensageiro , Transdução de Sinais
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