RESUMO
Antimicrobial peptides (AMPs), ancient scavengers of bacteria, are very poorly induced in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis), but the underlying mechanism remains unknown. Here, we report that L-alanine interacts with PRSS1 and unfreezes the inhibitory effect of PRSS1 on the activation of NF-κB pathway to induce the expression of AMPs, but mycobacterial alanine dehydrogenase (Ald) Rv2780 hydrolyzes L-alanine and reduces the level of L-alanine in macrophages, thereby suppressing the expression of AMPs to facilitate survival of mycobacteria. Mechanistically, PRSS1 associates with TAK1 and disruptes the formation of TAK1/TAB1 complex to inhibit TAK1-mediated activation of NF-κB pathway, but interaction of L-alanine with PRSS1, disables PRSS1-mediated impairment on TAK1/TAB1 complex formation, thereby triggering the activation of NF-κB pathway to induce expression of AMPs. Moreover, deletion of antimicrobial peptide gene ß-defensin 4 (Defb4) impairs the virulence by Rv2780 during infection in mice. Both L-alanine and the Rv2780 inhibitor, GWP-042, exhibits excellent inhibitory activity against M. tuberculosis infection in vivo. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses its own alanine dehydrogenase to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.
Assuntos
Alanina , Peptídeos Antimicrobianos , Macrófagos , Mycobacterium tuberculosis , NF-kappa B , Tuberculose , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/metabolismo , Animais , Camundongos , NF-kappa B/metabolismo , Humanos , Macrófagos/microbiologia , Macrófagos/metabolismo , Macrófagos/imunologia , Alanina/metabolismo , Peptídeos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/genética , Tuberculose/microbiologia , Tuberculose/imunologia , Alanina Desidrogenase/metabolismo , Alanina Desidrogenase/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transdução de Sinais , Camundongos Endogâmicos C57BL , Células RAW 264.7 , FemininoRESUMO
Internal N6-methyladenosine (m6A) modifications are among the most abundant modifications of messenger RNA, playing a critical role in diverse biological and pathological processes. However, the functional role and regulatory mechanism of m6A modifications in the immune response to Mycobacterium tuberculosis infection remains unknown. Here, we report that methyltransferase-like 14 (METTL14)-dependent m6A methylation of NAPDH oxidase 2 (Nox2) mRNA was crucial for the host immune defense against M. tuberculosis infection and that M. tuberculosis-secreted antigen EsxB (Rv3874) inhibited METTL14-dependent m6A methylation of Nox2 mRNA. Mechanistically, EsxB interacted with p38 MAP kinase and disrupted the association of TAB1 with p38, thus inhibiting the TAB1-mediated autophosphorylation of p38. Interaction of EsxB with p38 also impeded the binding of p38 with METTL14, thereby inhibiting the p38-mediated phosphorylation of METTL14 at Thr72. Inhibition of p38 by EsxB restrained liquid-liquid phase separation (LLPS) of METTL14 and its subsequent interaction with METTL3, preventing the m6A modification of Nox2 mRNA and its association with the m6A-binding protein IGF2BP1 to destabilize Nox2 mRNA, reduce ROS levels, and increase intracellular survival of M. tuberculosis. Moreover, deletion or mutation of the phosphorylation site on METTL14 impaired the inhibition of ROS level by EsxB and increased bacterial burden or histological damage in the lungs during infection in mice. These findings identify a previously unknown mechanism that M. tuberculosis employs to suppress host immunity, providing insights that may empower the development of effective immunomodulators that target M. tuberculosis.
RESUMO
TANK-binding kinase 1 (TBK1) is a nodal protein involved in multiple signal transduction pathways. In RNA virus-mediated innate immunity, TBK1 is recruited to the prion-like platform formed by MAVS and subsequently activates the transcription factors IRF3/7 and NF-κB to produce type I interferon (IFN) and proinflammatory cytokines for the signaling cascade. In this study, TRAF7 was identified as a negative regulator of innate immune signaling. TRAF7 interacts with TBK1 and promotes K48-linked polyubiquitination and degradation of TBK1 through its RING domain, impairing the activation of IRF3 and the production of IFN-ß. In addition, we found that the conserved cysteine residues at position 131 of TRAF7 are necessary for its function toward TBK1. Knockout of TRAF7 could facilitate the activation of IRF3 and increase the transcript levels of downstream antiviral genes. These data suggest that TRAF7 negatively regulates innate antiviral immunity by promoting the K48-linked ubiquitination of TBK1.
Assuntos
Interferon Tipo I , Transdução de Sinais , Humanos , Ubiquitinação , Imunidade Inata , Antivirais , Células HEK293 , Proteínas Serina-Treonina Quinases/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose TumoralRESUMO
The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Viroses , Animais , Humanos , Camundongos , Chaperonas Moleculares/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines in response to acute viral infection. However, excessive or prolonged production of IFN-I is harmful and even fatal to the host by causing autoimmune disorders. In this study, we identified mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) as a negative regulator in the RIG-I-like receptor (RLR) signaling pathway. MAP4K1, a member of Ste20-like serine/threonine kinases, was previously known as a prominent regulator in adaptive immunity by downregulating T-cell receptor (TCR) signaling and B-cell receptor (BCR) signaling. However, its role in regulating antiviral innate immune signaling is still unclear. This study reports an undiscovered role of MAP4K1, which inhibits RLR signaling by targeting TBK1/IKKε for proteasomal degradation via the ubiquitin ligase DTX4. We initially identify MAP4K1 as an interacting partner of TBK1 by yeast two-hybrid screens and subsequently investigate its function in RLR-mediated antiviral signaling pathways. Overexpression of MAP4K1 significantly inhibits RNA virus-triggered activation of IFN-ß and the production of proinflammatory cytokines. Consistently, knockdown or knockout experiments show opposite effects. Furthermore, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via DTX4. Knockdown of DTX4 abrogated the ubiquitination and degradation of TBK1/IKKε. Collectively, our results identify that MAP4K1 acts as a negative regulator in antiviral innate immunity by targeting TBK1/IKKε, discover a novel TBK1 inhibitor, and extend a novel functional role of MAP4K1 in immunity. IMPORTANCE TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines to restrict the spread of invading viruses. However, excessive or prolonged production of IFN-I is harmful to the host by causing autoimmune disorders. In this study, we identified that mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) is a negative regulator in the RLR signaling pathway. Notably, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via the ubiquitin ligase DTX4, leading to the negative regulation of the IFN signaling pathway. Previous studies showed that MAP4K1 has a pivotal function in adaptive immune responses. This study identifies that MAP4K1 also plays a vital role in innate immunity and outlines a novel mechanism by which the IFN signaling pathway is tightly controlled to avoid excessive inflammation. Our study documents a novel TBK1 inhibitor, which serves as a potential therapeutic target for autoimmune diseases, and elucidated a significant function for MAP4K1 linked to innate immunity in addition to subsequent adaptive immunity.
Assuntos
Citocinas/biossíntese , Quinase I-kappa B/metabolismo , Interferon beta/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Viroses/imunologia , Proteína DEAD-box 58/metabolismo , Humanos , Imunidade Inata/imunologia , Proteínas Serina-Treonina Quinases/genética , Vírus de RNA/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , UbiquitinaçãoRESUMO
The differentiation efficiency of adult stem cells undergoes a significant decline in aged animals, which is closely related to the decline in organ function and age-associated diseases. However, the underlying mechanisms that ultimately lead to this observed decline of the differentiation efficiency of stem cells remain largely unclear. This study investigated Drosophila midguts and identified an obvious upregulation of caudal (cad), which encodes a homeobox transcription factor. This factor is traditionally known as a central regulator of embryonic anterior-posterior body axis patterning. This study reports that depletion of cad in intestinal stem/progenitor cells promotes quiescent intestinal stem cells (ISCs) to become activate and produce enterocytes in the midgut under normal gut homeostasis conditions. However, overexpression of cad results in the failure of ISC differentiation and intestinal epithelial regeneration after injury. Moreover, this study suggests that cad prevents intestinal stem/progenitor cell differentiation by modulating the Janus kinase/signal transducers and activators of the transcription pathway and Sox21a-GATAe signaling cascade. Importantly, the reduction of cad expression in intestinal stem/progenitor cells restrained age-associated gut hyperplasia in Drosophila. This study identified a function of the homeobox gene cad in the modulation of adult stem cell differentiation and suggested a potential gene target for the treatment of age-related diseases induced by age-related stem cell dysfunction.
Assuntos
Células-Tronco Adultas/metabolismo , Diferenciação Celular/genética , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/fisiologia , Fatores Etários , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Intestinos/citologia , Janus Quinases/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Retinoic acid-inducible gene I (RIG-I) plays a critical role in the recognition of intracytoplasmic viral RNA. Upon binding to the RNA of invading viruses, the activated RIG-I translocates to mitochondria, where it recruits adapter protein MAVS, causing a series of signaling cascades. In this study, we demonstrated that Hsp70 binding protein 1 (HSPBP1) promotes RIG-I-mediated signal transduction. The overexpression of HSPBP1 can increase the stability of RIG-I protein by inhibiting its K48-linked ubiquitination, and promote the activation of IRF3 and the production of IFN-ß induced by Sendai virus. Knockdown and knockout of HSPBP1 leads to down-regulation of virus-induced RIG-I expression, inhibits IRF3 activation, and reduces the production of IFNB1. These results indicate that HSPBP1 positively regulates the antiviral signal pathway induced by inhibiting the K48-linked ubiquitination of RIG-I.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína DEAD-box 58/metabolismo , Imunidade Inata/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Receptores Imunológicos/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , UbiquitinaçãoRESUMO
Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous ß-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified to be a ß-D-(1-3)-linked glucan backbone with a single ß-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.
Assuntos
Grifola/química , Lectinas Tipo C/metabolismo , Oligonucleotídeos/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , beta-Glucanas/química , Agaricales/química , Animais , Citocinas/metabolismo , Camundongos , Conformação Molecular , Poli A , Células RAW 264.7RESUMO
Retinoic acid-inducible gene-I (RIG-I) belongs to the RIGI-like receptors (RLRs), a class of primary pattern recognition receptors. It senses viral double-strand RNA in the cytoplasm and delivers the activated signal to its adaptor virus-induced signaling adapter (VISA), which then recruits the downstream TNF receptor-associated factors and kinases, triggering a downstream signal cascade that leads to the production of proinflammatory cytokines and antiviral interferons (IFNs). However, the mechanism of RIG-I-mediated antiviral signaling is not fully understood. Here, we demonstrate that chitinase domain-containing 1 (CHID1), a member of the chitinase family, positively regulates the RLR antiviral signaling pathway by targeting the RIG-I/VISA signalosome. CHID1 overexpression enhances the activation of nuclear factor κB (NF-кB) and interferon regulatory factor 3 (IRF3) triggered by Sendai virus (SeV) by promoting the polyubiquitination of RIG-I and VISA, thereby potentiating IFN-ß production. CHID1 knockdown in human 239T cells inhibits SeV-induced activation of IRF3 and NF-κB and the induction of IFN-ß. These results indicate that CHID1 positively regulates RLR antiviral signal, revealing the novel mechanism of the RIG-I antiviral signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteína DEAD-box 58/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Proteínas de Transporte/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Interferon beta/biossíntese , Proteoma , Proteômica/métodos , Receptores Imunológicos , UbiquitinaçãoRESUMO
A robust and sensitive method of solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and performed for the simultaneous determination of eleven aminoglycosides (AGs) in royal jelly and honey. After sample extraction by a phosphate buffer containing trichloroacetic acid (TCA) and ethylenediaminetetracetic acid disodium salt (Na2EDTA), the extraction solution was subjected to a parallel solid-phase extraction for clean-up prior to the LC-MS/MS analysis. The same method was applied to analyze two completely different matrices, honey and royal jelly. Good sensitivity, repeatability, and recovery were obtained by using the mobile phase without an ion-pairing reagent such as heptafluorobutyric acid (HFBA) or sodium heptanesulfonate. The calibration curves of the honey and royal jelly samples exhibited a good linear response (R2â¯>â¯0.99) at six concentrations in the range of 10-1000⯵g/mL. The limit of quantification (LOQ) of the AGs ranged from 10 to 25⯵g/kg in the honey and from 12.5 to 25⯵g/kg in the royal jelly. The recoveries of the AGs for the honey and royal jelly samples were in the range of 79.48% to 108.95% and 74.61% to 113.70% respectively and the relative standard deviations (RSDs) were between 1.23% and 9.59%, and between 1.51% and 9.98%, respectively. The proposed approach has been allowed in China as a reference method for the simultaneous determination of eleven AGs in honey and royal jelly.
Assuntos
Aminoglicosídeos/análise , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/química , Análise de Alimentos/métodos , Mel/análise , Extração em Fase Sólida , Espectrometria de Massas em Tandem , China , Limite de DetecçãoRESUMO
Several different extraction procedures including perchloric acid extraction, boiling water extraction and boiling magnesium sulfate solution extraction were studied for the extraction of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) from the royal jelly. Among these methods, the extraction with 5% perchloric acid at below 4 degrees C was the optimum extraction method. A simple, fast and sensitive ultra-performance liquid chromatographic (UPLC) method was developed for the determination of ATP, ADP and AMP in royal jelly. The separation was achieved within 4 min using a BEH Shield RP18 column (100 mm x 2.1 mm, 1.7 microm) with 50 mmol/L monoammonium phosphate solution (pH 6.5) and acetonitrile as the mobile phase. The spiked recoveries of ATP, ADP and AMP were 84.1% -94.3%, 86.2% -93.7% and 91.0% -104.3%, respectively. The relative standard deviations were less than 10%. This method was successfully applied to the analysis of some royal jelly samples from beekeepers and markets for the investigation of distribution of ATP, ADP and AMP in royal jelly samples.
Assuntos
Nucleotídeos de Adenina/análise , Nucleotídeos de Adenina/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/química , Temperatura Alta , Limite de Detecção , Modelos Lineares , Sulfato de Magnésio/química , Água/químicaRESUMO
Polysaccharides in Several traditional Chinese medicine compounds of Dioscorea bulbifera L, Dioscorea bulbifera L and Chinese Angelica compounds (3: 2 by mass ratio), Hedyotis diffusa Willd and Scutellrla barbata D. Don compounds (3: 2 by mass ratio) were extracted by ultrasound and the content of polysaccharides were determined by the colorimetry. Dioscorea bulbifera L and Chinese Angelica compounds contained the biggest polysaccharide, 16. 509%. All of these three kinds of polysaccharide had anti-tumor activity. The anti-tumor activity was Dioscorea bulbifera L and Chinese Angelica compounds > Hedyotis diffusa Willd and Scutellrla barbata D. Don compounds > Dioscorea bubifera L.