Regulation of TLR4 signaling through the TRAF6/sNASP axis by reversible phosphorylation mediated by CK2 and PP4

2021 ◽  
Vol 118 (47) ◽  
pp. e2107044118
Author(s):  
Feng-Ming Yang ◽  
Hui-Ming Chang ◽  
Edward T. H. Yeh
Keyword(s):  
Innate Immunity ◽  
Signaling Pathways ◽  
Recombinant Adenovirus ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Gene Encoding ◽  
Downstream Signaling ◽  
Signaling Complex ◽  
Nuclear Autoantigenic Sperm Protein ◽  
Tlr4 Activation

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor–associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor–associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4–infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.

scholarly journals Multiple environmental signaling pathways control the differentiation of RORγt-expressing regulatory T cells

2019 ◽  
Author(s):  
Hind Hussein ◽  
Sébastien Denanglaire ◽  
Frédéric Van Gool ◽  
Abdulkader Azouz ◽  
Yousra Ajouaou ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Ectopic Expression ◽  
Critical Factor ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Effect ◽  
Commensal Microbiota ◽  
Negative Effect ◽  
Treg Differentiation ◽  
Treg Development

AbstractRORγt-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of RORγt expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of RORγt+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that RORγt+ Treg development is controlled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-β-derived signals act in favor of RORγt+ Treg development. Ectopic expression of c-Maf did not rescue RORγt expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-γ/STAT1 signaling pathway acts as a negative regulator of RORγt+ Treg differentiation in a c-Maf independent fashion.These data thus argue for a complex integrative signaling network that finely tunes RORγt expression in Tregs. The finding that type 1 inflammation impedes RORγt+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.

HSV ICP0 Inhibits TLR-Mediated NF-κB Response to TLR Signaling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5487-5487
Author(s):  
Khaled A. Tolba ◽  
William Bowers ◽  
Yaohong Tan ◽  
Sandrine Daubeuf ◽  
Howard J. Federoff ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Signaling Pathways ◽  
E3 Ligase ◽  
Ring Domain ◽  
Negative Regulator ◽  
Expression Vectors ◽  
Tlr Signaling ◽  
Ubiquitin E3 Ligase ◽  
Early Protein ◽  
Ligase Function

Abstract HSV infection activates a robust innate response through engagement of multiple pattern recognition receptors (PRR) including both TLR (TLR2 and TLR9) as well as non-TLR. Signaling events downstream of these receptors activate NF-κB and IRF3 responsive genes and initiate an innate inflammatory response aimed at controlling viral replication and spread. In this work, we studied immune suppressive activity of a replication-defective HSV virus and identified the immediate early protein ICP0 as a negative regulator of both NF-κB and IRF3 signaling. ICP0 possesses an ubiquitin E3 ligase function through its NH2 RING domain, as well as de-ubiquitinating activity through its association with the cellular de-ubiquitinating enzyme USP7 (HAUSP). We show that these two domains of ICP0 function independently to suppress IRF3 and NF-κB signaling, respectively and, in the process, effectively shut down host innate immunity to HSV infection. Although ICP0 inhibition of IRF3 has been reported, inhibition of TLR-mediated NF-κB response has not been previously described. We show that ICP0 globally inhibits NF-κB response to all TLR receptors as well as IL-1R. ICP0 exerts this activity by associating with USP7 and altering its cellular localization from a nuclear to cytoplasmic protein. In the cytosol, USP7 associates with and de-ubiquitinates TRAF6 and IKK-γ (NEMO), two signaling components of the TLR-mediated NF-κB pathway that are poly-ubiquitinated upon TLR activation. ICP-0 expression vectors harboring point mutations/deletions that target the RING domain E3 ligase function or compromise ICP-0 ability to bind USP7 would selectively inhibit its ability to interfere with either IRF3 or NF-kB signaling, respectively. In support of this, knockdown of endogenous USP7 by RNAi severely impaired ICP0-mediated inhibition of NF-κB response while leaving its capacity to inhibit IRF3 intact. In contrast, over-expression of USP-7 was sufficient to inhibit TLR-mediated NF-κB response. Ability of ICP-0 to inhibit both IRF3 and NF-κB signaling pathways, the former through its E3 ligase function and the latter through its association with USP-7, affords HSV comprehensive protection from host immunity during repeated cycles of lytic infection and reactivation from latency. The work also identifies a rare example of how two seemingly contradictory biologic functions resident within ICP0, namely ubiquitin E3 ligase activity at the NH2 terminus RING domain and de-ubiquitinating activity through association with USP-7, could cooperate to inhibit multiple signaling pathways necessary for efficient silencing of innate immunity. Finally, the work also identifies a previously unknown function for USP7 in regulating innate signaling beyond its known function as a regulator of p53.

scholarly journals SOCS3 Drives Proteasomal Degradation of TBK1 and Negatively Regulates Antiviral Innate Immunity

2015 ◽  
Vol 35 (14) ◽  
pp. 2400-2413 ◽  
Author(s):  
Dong Liu ◽  
Chunjie Sheng ◽  
Shijuan Gao ◽  
Chen Yao ◽  
Jiandong Li ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Influenza A ◽  
Critical Role ◽  
Antiviral Agents ◽  
Ectopic Expression ◽  
Proteasomal Degradation ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Type I ◽  
Antiviral Innate Immunity

TANK-binding kinase 1 (TBK1)-mediated induction of type I interferon (IFN) plays a critical role in host antiviral responses and immune homeostasis. The negative regulation of TBK1 activity is largely unknown. We report that suppressor of cytokine signaling 3 (SOCS3) inhibits the IFN-β signaling pathway by promoting proteasomal degradation of TBK1. Overexpression and knockdown experiments indicated that SOCS3 is a negative regulator of IFN regulatory factor 3 (IRF3) phosphorylation and IFN-β transcription. Moreover, SOCS3 directly associates with TBK1, and they colocalize in the cytoplasm. SOCS3 catalyzes K48-linked polyubiquitination of TBK1 at Lys341 and Lys344 and promotes subsequent TBK1 degradation. On the contrary, SOCS3 knockdown markedly increases the abundance of TBK1. Interestingly, both the BOX domain of SOCS3 and Ser172 phosphorylation of TBK1 are indispensable for the processes of ubiquitination and degradation. Ectopic expression of SOCS3 significantly inhibits vesicular stomatitis virus (VSV) and influenza A virus strain A/WSN/33 (WSN)-induced IRF3 phosphorylation and facilitates the replication of WSN virus by detecting the transcription of its viral RNA (vRNA). Knockdown of SOCS3 represses WSN replication. Collectively, these results demonstrate that SOCS3 acts as a negative regulator of IFN-β signal by ubiquitinating and degrading TBK1, shed light on the understanding of antiviral innate immunity, and provide a potential target for developing antiviral agents.

scholarly journals Homeostatic regulation of STING by retrograde membrane traffic to the ER

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kojiro Mukai ◽  
Emari Ogawa ◽  
Rei Uematsu ◽  
Yoshihiko Kuchitsu ◽  
Fumika Kiku ◽  
...  
Keyword(s):  
Resting State ◽  
Complex I ◽  
Membrane Traffic ◽  
Retrograde Transport ◽  
Homeostatic Regulation ◽  
Gene Encoding ◽  
Downstream Signaling ◽  
Intermediate Compartment ◽  
Downstream Signaling Pathway ◽  
Copa Syndrome

AbstractCoat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.

scholarly journals Orai–vascular endothelial-cadherin signaling complex regulates high-glucose exposure-induced increased permeability of mouse aortic endothelial cells

2021 ◽  
Vol 9 (1) ◽  
pp. e002085
Author(s):  
Yuan Wei ◽  
Suwen Bai ◽  
YanHeng Yao ◽  
Wenxuan Hou ◽  
Junwei Zhu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Membrane ◽  
High Glucose ◽  
Expression Level ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Downstream Signaling ◽  
Signaling Complex ◽  
Aortic Endothelium ◽  
Aortic Endothelial

IntroductionDiabetes-associated endothelial barrier function impairment might be linked to disturbances in Ca2+ homeostasis. To study the role and molecular mechanism of Orais–vascular endothelial (VE)-cadherin signaling complex and its downstream signaling pathway in diabetic endothelial injury using mouse aortic endothelial cells (MAECs).Research design and methodsThe activity of store-operated Ca2+ entry (SOCE) was detected by calcium imaging after 7 days of high-glucose (HG) or normal-glucose (NG) exposure, the expression levels of Orais after HG treatment was detected by western blot analysis. The effect of HG exposure on the expression of phosphorylated (p)-VE-cadherin and VE-cadherin on cell membrane was observed by immunofluorescence assay. HG-induced transendothelial electrical resistance was examined in vitro after MAECs were cultured in HG medium. FD-20 permeability was tested in monolayer aortic endothelial cells through transwell permeability assay. The interactions between Orais and VE-cadherin were detected by co-immunoprecipitation and immunofluorescence technologies. Immunohistochemical experiment was used to detect the expression changes of Orais, VE-cadherin and p-VE-cadherin in aortic endothelium of mice with diabetes.Results(1) The expression levels of Orais and activity of SOCE were significantly increased in MAECs cultured in HG for 7 days. (2) In MAECs cultured in HG for 7 days, the ratio of p-VE-cadherin to VE-cadherin expressed on the cell membrane and the FD-20 permeability in monolayer endothelial cells increased, indicating that intercellular permeability increased. (3) Orais and VE-cadherin can interact and enhance the interaction ratio through HG stimulation. (4) In MAECs cultured with HG, the SOCE activator ATP enhanced the expression level of p-VE-cadherin, and the SOCE inhibitor BTP2 decreased the expression level of p-VE-cadherin. (5) Significantly increased expression of p-VE-cadherin and Orais in the aortic endothelium of mice with diabetes.ConclusionHG exposure stimulated increased expression of Orais in endothelial cells, and increased VE-cadherin phosphorylation through Orais–VE-cadherin complex and a series of downstream signaling pathways, resulting in disruption of endothelial cell junctions and initiation of atherosclerosis.

scholarly journals Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

2020 ◽  
Vol 9 (1) ◽  
pp. 71
Author(s):  
Julia Marente ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Wild Strain ◽  
Ring Finger ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Fusarium Fujikuroi ◽  
Structural Genes ◽  
Gene Encoding ◽  
In The Wild ◽  
Set Up

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

scholarly journals Immunoregulatory Property of C-Type Lectin-Like Receptors in Fibrosing Interstitial Lung Diseases

2020 ◽  
Vol 21 (10) ◽  
pp. 3665
Author(s):  
Wiwin Is Effendi ◽  
Tatsuya Nagano ◽  
Helmia Hasan ◽  
Resti Yudhawati
Keyword(s):  
Immune System ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Lung Diseases ◽  
Wound Repair ◽  
Tissue Injury ◽  
Interstitial Lung Diseases ◽  
Downstream Signaling ◽  
Cytokine Activation ◽  
Immune Impairment

The innate immune system identifies exogenous threats or endogenous stress through germline-encoded receptors called pattern recognition receptors (PRRs) that initiate consecutive downstream signaling pathways to control immune responses. However, the contribution of the immune system and inflammation to fibrosing interstitial lung diseases (ILD) remains poorly understood. Immunoreceptor tyrosine-based motif-bearing C-type lectin-like receptors (CTLRs) may interact with various immune cells during tissue injury and wound repair processes. Dectin-1 is a CTLR with dominant mechanisms manifested through its intracellular signaling cascades, which regulate fibrosis-promoting properties through gene transcription and cytokine activation. Additionally, immune impairment in ILD facilitates microbiome colonization; hence, Dectin-1 is the master protector in host pulmonary defense against fungal invasion. Recent progress in determining the signaling pathways that control the balance of fibrosis has implicated immunoreceptor tyrosine-based motif-bearing CTLRs as being involved, either directly or indirectly, in the pathogenesis of fibrosing ILD.

Innate Immunity Signaling Pathways: Links between Immunonutrition and Responses to Sepsis

2011 ◽  
Vol 59 (2) ◽  
pp. 139-150 ◽  
Author(s):  
Robert Słotwiński ◽  
Sylwia Słotwińska ◽  
Sylwia Kędziora ◽  
Barbara-Joanna Bałan
Keyword(s):  
Innate Immunity ◽  
Signaling Pathways

Candida tropicalis induces pro-inflammatory cytokine production, NF-κB and MAPKs pathways regulation, and dectin-1 activation

2018 ◽  
Vol 64 (12) ◽  
pp. 937-944 ◽  
Author(s):  
Zhimin Duan ◽  
Qing Chen ◽  
Rong Zeng ◽  
Leilei Du ◽  
Caixia Liu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Cytokine ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Downstream Signaling ◽  
Tnf Α ◽  
Mitogen Activated Protein

The prevalence of Candida infection induced by non-albicans Candida (NAC) species is increasing. However, as a common NAC species, C. tropicalis has received much less study in terms of host immunity than C. albicans has. In this study, we evaluated the pro-inflammatory cytokine responses evoked by C. tropicalis and determined whether dectin-1 and downstream NF-κB and mitogen-activated protein kinases (MAPKs) signaling pathways played roles in inflammation in human peripheral blood mononuclear cells (PBMCs) and THP-1 macrophage-like cells. Exposure of PBMCs and THP-1 macrophage-like cells to C. tropicalis led to the enhanced gene expression and secretion of TNF-α and IL-6 in a time- and dose-dependent manner. THP-1 macrophage-like cells being challenged by C. tropicalis resulted in the activation of the NF-κB, p38, and ERK1/2 MAPK signaling pathways. We also found that the expression of dectin-1 was increased with C. tropicalis treatment. These data reveal that dectin-1 may play a role in sensing the inflammation response induced by C. tropicalis and that NF-κB and MAPK are involved in the downstream signaling pathways in macrophages.

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