Receptor Domain-containing Adaptor Research Articles

HDAC5 enhances IRF3 activation and is targeted for degradation by protein C6 from orthopoxviruses including Monkeypox virus and Variola virus

Histone deacetylases (HDACs) regulate gene expression and innate immunity. Previously, we showed that HDAC5 is degraded during Vaccinia virus (VACV) infection and is a restriction factor for VACV and herpes simplex virus type 1. Here, we report that HDAC5 promotes interferon regulatory factor 3 (IRF3) activation downstream of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 or Sendai virus-mediated stimulation without requiring HDAC activity. Loss of HDAC5-mediated IRF3 activation is restored by re-introduction of HDAC5 but not HDAC1 or HDAC4. The antiviral activity of HDAC5 is antagonized by VACV protein C6 and orthologs from the orthopoxviruses cowpox, rabbitpox, camelpox, monkeypox, and variola. Infection by many of these viruses induces proteasomal degradation of HDAC5, and expression of C6 alone can induce HDAC5 degradation. Mechanistically, C6 binds to the dimerization domain of HDAC5 and prevents homodimerization and heterodimerization with HDAC4. Overall, this study describes HDAC5 as a positive regulator of IRF3 activation and provides mechanistic insight into how the poxviral protein C6 binds to HDAC5 to antagonize its function.

A novel PD-1/PD-L1 pathway-related seven-gene signature for the development and validation of the prognosis prediction model for breast cancer.

Breast cancer (BC/BRCA) is the most common carcinoma in women. The average 5-year survival rate of BC patients with stage IV disease is 26%. A considerable proportion of patients still do not receive effective therapy. It is an unmet need to identify novel biomarkers for BC patients. Herein, we evaluated whether the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) status is associated with the clinical outcomes of BC, based on data from The Cancer Genome Atlas (TCGA). Clinical and transcriptome data of BC patients were obtained from TCGA dataset, and prognostic genes in BC patients were identified, as well as the PD-1/PD-L1 pathway mainly associating with the BC patients. Following the execution of the consensus clustering algorithm, BC patients were segregated into two clusters, and subsequent investigation of the potential mechanisms between them was carried out. A comparison of ferroptosis and N6-methyladenosine (m6A) was conducted between the two groups with the greatest difference in prognosis. Based on least absolute shrinkage and selection operator (LASSO) analysis, a signature associated with the PD-1/PD-L1 pathway was developed, and the prognosis outcome and the predictive accuracy of the signature model were further assessed. Prognostic genes in BC patients were studied using TCGA data and it was found that the PD-1/PD-L1 pathway was most associated with the BC patients. Then, a low-risk (C1) group and a high-risk (C2) group of BC patients were constructed based on a PD-1/PD-L1 pathway-related signature. The functional analyses suggested that the underlying mechanisms between these groups were mainly associated with immune-related pathways. We found that ferroptosis and m6A were significantly different between the two groups. A PD-1/PD-L1 pathway-related gene signature was further developed to predict survival of BC patients, including 7 genes [mitogen-activated protein kinase kinase 6 (MAP2K6), NF-kappa-B inhibitor alpha (NFKBIA), NFKB Inhibitor Epsilon (NFKBIE), Interferon gamma (IFNG), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), IkappaB kinase (CHUK), and Casein kinase 2 alpha 3 gene (CSNK2A3)]. The receiver operating characteristic (ROC) curves were analyzed to further assess the prognostic values of these 7 genes. The 1-, 3-, and 5-year values of the areas under the curve (AUCs) for overall survival were 0.651, 0.658, and 0.653 in this seven gene signature model, respectively. PD-1/PD-L1 pathway-related subtypes of BC were identified, which were closely associated with the immune microenvironment, the ferroptosis status, and m6A in BC patients. The gene signature involved in the PD-1/PD-L1 pathway might help to make a distinction and predict prognosis in BC patients.

Effectiveness of Narciclasine in Suppressing the Inflammatory Response in Sepsis: Molecular Docking and In Silico Studies.

Narciclasine is an alkaloid belonging to the Amaryllidaceae family which has been reported to have many beneficial properties. Especially its anticancer properties have been widely reported. Here, we have focused on its potential use in suppressing the inflammatory response in sepsis using in silico methods. Lipopolysaccharide (LPS) is an endotoxin which is present in the outer membrane of gram-negative bacteria and is a crucial player in the pathogenesis of gram-negative sepsis. Activation of toll-like receptor 4 (TLR4) signaling by LPS is an important event in the pathogenesis of gram-negative sepsis. This initiates a downstream signaling pathway comprising of several adaptor proteins such as toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase (IRAK)-1, IRAK-4, interferon regulatory factor 3 (IRF-3), tumor necrosis factor receptor-associated factor 6 (TRAF-6) leading to nuclear factor kappa B (NF-κβ) activation resulting in elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. S100 calcium binding proteins A8/A9 (S100A8/A9) have been found to be an agonist of TLR4, and it amplifies the inflammatory response in sepsis. Molecular docking studies of narciclasine with target proteins associated with the LPS-TLR4 pathway showed that it has good binding affinity and stable interactions with the targets studied. Molecular dynamics (MD) simulation studies over 100 ns showed that most of the ligand-target complexes were stable. The structures of all the targets except TRAF-6 were retrieved from the Protein Data Bank (PDB) database. Homology modeling was done to predict the 3-dimensional structure of TRAF-6. MD simulation of narciclasine-TRAF-6 complex showed that the structure is stable. Metapocket was used for active site prediction in the target proteins. Toxicity analysis by admetSAR revealed that narciclasine was readily biodegradable and exhibited minimum toxicity. These results indicate that narciclasine has effective anti-inflammatory properties which could be useful in suppressing the inflammatory response in sepsis.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms’ expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-β expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-β signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-β, signaling.

Inhibition of HMGB1 Attenuates Spinal Cord Edema by Reducing the Expression of Na+-K+-Cl- Cotransporter-1 and Na+/H+ Exchanger-1 in Both Astrocytes and Endothelial Cells After Spinal Cord Injury in Rats.

Sodium/water transport through Na+-K+-Cl- cotransporter-1 (NKCC1) and sodium/hydrogen exchanger-1 (NHE1) in both astrocytes and endothelial cells is critical to cytotoxic and ionic edema following spinal cord injury (SCI). High-mobility group box-1 (HMGB1) promotes spinal cord edema after SCI. Accordingly, we sought to identify both the role of HMGB1 and the mechanism of its effect on NKCC1 and NHE1 expression in astrocytes and endothelial cells as well as the role of the regulation of spinal cord edema after SCI. An SCI model was generated in adult female rats using a heavy falling object, and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was generated in rat spinal cord astrocytes and microvascular endothelial cells. The inhibition of HMGB1 reduced NKCC1 and NHE1 expression in the spinal cord of SCI rats, in cultured spinal cord astrocytes, and in cultured microvascular endothelial cells. The effects of HMGB1 on NKCC1 and NHE1 expression were mediated-at least in part-by activation of the Toll-like receptor 4 (TLR4)-Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-nuclear factor-kappa B (NF-κB) signaling pathway. The inhibition of NKCC1 or NHE1 decreased the spinal cord water content in rats following SCI, increased the Na+ concentration in the medium of cultured astrocytes after OGD/R, and reduced the astrocytic cell volume and AQP4 expression. These results imply that HMGB1 inhibition results in a reduction in NKCC1 and NHE1 expression in both astrocytes and microvascular endothelial cells and thus decreases spinal cord edema after SCI in rats and that these effects occur through the HMGB1-TLR4-TRIF-NF-κB signaling pathway.

TLR5-Derived, TIR-Interacting Decoy Peptides to Inhibit TLR Signaling.

TLR5, which is activated by flagellin, plays an important role in initiating immune response to a broad spectrum of motile bacterial pathogens. TLRs induce intracellular signaling via dimerization of their TIR domains followed by adapter recruitment through multiple interactions of receptor and adapter TIRs. Here, a library of cell-permeable decoy peptides derived from the TLR5 TIR was screened for TLR5 signaling inhibition in the HEK-Blue-mTLR5 reporter cell line. The peptide demonstrating the strongest inhibition, 5R667, corresponded to the second helix of the region between the third and fourth β-strands (helix C″). In addition to the TLR5-induced cytokine expression, 5R667 inhibited cytokine expression elicited by TLR4, TLR2, and TLR9. 5R667 also suppressed the systemic cytokine induction elicited by LPS administration in mice. 5R667 binding specificity was studied by time-resolved fluorescence spectroscopy in a cell-based assay. 5R667 demonstrated a multispecific binding pattern with respect to TIR domains: It bound TIRs of TLR adapters of the MyD88-dependent pathway, Toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP) and MyD88, and also the TIR of TLR5. TR667, the peptide derived from the TIRAP region, which is structurally homologous to 5R667, demonstrated binding and inhibitory properties similar to that of 5R667. The surface-exposed residues within TIR regions represented by 5R667 and TR667 form motifs, which are nearly 90% conserved in vertebrate evolution and are distinctive of TLR5 and TIRAP TIR domains. Thus, we have identified an evolutionary conserved adapter recruitment motif within TLR5 TIR, the function of which can be inhibited by selective cell-permeable decoy peptides, which can serve as pan-specific TLR inhibitors.

Prothymosin α Plays Role as a Brain Guardian through Ecto-F1 ATPase-P2Y12 Complex and TLR4/MD2.

Prothymosin alpha (ProTα) was discovered to be a necrosis inhibitor from the conditioned medium of a primary culture of rat cortical neurons under starved conditions. This protein carries out a neuronal cell-death-mode switch from necrosis to apoptosis, which is, in turn, suppressed by a variety of neurotrophic factors (NTFs). This type of NTF-assisted survival action of ProTα is reproduced in cerebral and retinal ischemia-reperfusion models. Further studies that used a retinal ischemia-reperfusion model revealed that ProTα protects retinal cells via ecto-F1 ATPase coupled with the Gi-coupled P2Y12 receptor and Toll-like receptor 4 (TLR4)/MD2 coupled with a Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF). In cerebral ischemia-reperfusion models, ProTα has additional survival mechanisms via an inhibition of matrix metalloproteases in microglia and vascular endothelial cells. Heterozygous or conditional ProTα knockout mice show phenotypes of anxiety, memory learning impairment, and a loss of neurogenesis. There are many reports that ProTα has multiple intracellular functions for cell survival and proliferation through a variety of protein-protein interactions. Overall, it is suggested that ProTα plays a key role as a brain guardian against ischemia stress through a cell-death-mode switch assisted by NTFs and a role of neurogenesis.

Associations among SNPs in two addictive genes, food addiction, and antioxidant markers in recreationally active young women.

Introduction: food addiction is associated with genetic polymorphisms and decreased antioxidant intake. Objectives: this study determined the associations among food addiction, dopamine receptor 2 (DRD2) and toll-interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP rs625413) gene polymorphisms, antioxidant capacities, and zinc levels among recreationally active Turkish women. Methods: the Yale Food Addiction Scale was used to evaluate the food addiction status. Serum antioxidant capacities and zinc levels were evaluated by blood analyses. Deoxyribonucleic acid (DNA) extraction was performed using peripheral blood leukocytes, and the polymorphism status of the DRD2 Taq 1A and TIRAP genes was investigated using a commercial kit. Results: the frequencies of the heterozygous genotypes of DRD2 Taq 1A and TIRAP were 23.1 % and 31.4 %, respectively, and the frequency of risk allele homozygous genotypes was 3.2 %. Most participants (94.4 %) had a nonpolymorphic/wild (CC) genotype in both genes; however, 11.5 % of the participants had a food addiction. The differences between serum antioxidant capacities, zinc levels, and body mass indices of those with and without food addiction were statistically significant. However, there were no differences in the serum zinc and antioxidant levels among the different genotypes. Conclusion: food addiction in young Turkish women was not associated with DRD2 Taq 1A or TIRAP polymorphisms but was associated with serum antioxidant capacities and zinc levels. Further studies on different loci of the same genes or genotypes of different genes with larger sample sizes are warranted.

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

A genome-wide search of Toll/Interleukin-1 receptor (TIR) domain-containing adapter molecule (TICAM) and their evolutionary divergence from other TIR domain containing proteins

Toll/Interleukin-1 receptor (TIR) domains are cytoplasmic domain that mediates receptor signalling. These domains are present in proteins like Toll-like receptors (TLR), its signaling adaptors and Interleukins, that form a major part of the immune system. These TIR domain containing signaling adaptors binds to the TLRs and interacts with their TIR domains for downstream signaling. We have examined the evolutionary divergence across the tree of life of two of these TIR domain containing adaptor molecules (TICAM) i.e., TIR domain-containing adapter-inducing interferon-β (TRIF/TICAM1) and TIR domain containing adaptor molecule2 (TRAM/TICAM2), by using computational approaches. We studied their orthologs, domain architecture, conserved motifs, and amino acid variations. Our study also adds a timeframe to infer the duplication of TICAM protein from Leptocardii and later divergence into TICAM1/TRIF and TICAM2/TRAM. More evidence of TRIF proteins was seen, but the absence of conserved co-existing domains such as TRIF-NTD, TIR, and RHIM domains in distant relatives hints on diversification and adaptation to different biological functions. TRAM was lost in Actinopteri and has conserved domain architecture of TIR across species except in Aves. An additional isoform of TRAM, TAG (TRAM adaptor with the GOLD domain), could be identified in species in the Mesozoic era. Finally, the Hypothesis based Likelihood ratio test was applied to look for selection pressure amongst orthologues of TRIF and TRAM to search for positively selected sites. These residues were mostly seen in the non-structural region of the proteins. Overall, this study unravels evolutionary information on the adaptors TRAM and TRIF and how well they had duplicated to perform diverse functions by changes in their domain architecture across lineages.

Effects of acupuncture on microglial polarization and the TLR4/TRIF/MyD88 pathway in a rat model of traumatic brain injury.

Neuroinflammation caused by traumatic brain injury (TBI) can lead to neurological deficits. Acupuncture can inhibit neuroinflammation and promote nerve repair; however, the specific mechanism is still unclear. The purpose of this study was to explore whether acupuncture could modulate the M1 and M2 phenotypic polarization of microglia in a rat model of TBI via the toll-like receptor 4 (TLR4)/intracellular toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. A total of 90 adult male Sprague-Dawley (SD) rats, SPF grade, were randomly divided into a normal group, model group and acupuncture group. Each group was further divided into three subgroups (first, third, and fifth day groups) according to the treatment time (n = 10 rats/subgroup). We used the modified neurological severity score (mNSS) method to quantify neurological deficits before and after modeling. We used Nissl staining to observe the pathological changes in brain tissue, flow cytometry to detect the proportion of M1 and M2 polarized microglia in the injured area on the first, third and fifth day, and co-immunoprecipitation (Co-IP) to examine TLR4/TRIF/MyD88 expression in microglia on the first, third and fifth day, as well as expression of the amount of binding of TLR4 with TRIF and MyD88. Compared to the model group, mNSS in the acupuncture group gradually decreased and pathological morphology improved. The proportion of CD11b/CD86 positive cells was decreased, while that of CD11b/CD206 was increased in the acupuncture group. Expression of IP TLR4, IP TRIF and IP MyD88 also decreased in the acupuncture group. The results of this study demonstrate that one of the mechanisms through which acupuncture mitigates neuroinflammation and promotes nerve repair in TBI rats may be inhibition of M1 phenotypic polarization and promotion of M2 phenotypic polarization through inhibition of the TLR4/TRIF/MyD88 signaling pathway.

SARM suppresses glioma progression in GL261 glioma cells and regulates microglial polarization

Microglia is the major cellular component of glioma mass that promotes glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Sterile alpha and HEAT/Armadillo motif (SARM), a member of the Toll-interleukin-1 receptor (TIR) domain-containing adaptor family, is primarily expressed in the central nervous system. However, the role of SARM in glioma is still undefined. In the present work, we examined the function of SARM in microglial polarization and glioma progression. Our results showed that forced the expression of SARM in GL261 glioma cells inhibited tumor growth, and reduced interleukin (IL)-6 secretion in conditioned media. Silencing of SARM in microglia cells inhibited IL-4-induced M2 polarization, enhanced lipopolysaccharide -induced M1 microglial polarization. Furthermore, overexpression of SARM increased the migration of microglia cells upon TGFβ stimulation. These data suggested that SARM is involved in neuro-inflammation and microglia activation. In summary, this study provides novel insight into the mechanisms of microglial polarization.

TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

TIRAP-mediated activation of p38 MAPK in inflammatory signaling

The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its discovery in 2001 due to its dynamic nature and subcellular localization to regulate multiple signaling through several protein–protein interactions (PPIs). Structural analysis of these interactions can reveal a better understanding of their conformational dynamics and the nature of their binding. Tyrosine phosphorylation in the TIR domain of TIRAP is very critical for its function. In toll-like receptor (TLR) 4/2 signalling, Bruton's tyrosine kinase (BTK) and Protein kinase C delta (PKCδ) are known to phosphorylate the Y86, Y106, Y159, and Y187 of TIRAP which is crucial for the downstream function of MAPKs (mitogen-activated protein kinases) activation. The objective of this study is to understand the interaction of TIRAP with p38 MAPK through molecular docking and identify the importance of TIRAP tyrosine phosphorylation in p38 MAPK interaction. In this structural study, we performed an in-silico molecular docking using HADDOCK 2.4, pyDockWEB, ClusPro 2.0, and ZDOCK 3.0.2 tools to unravel the interaction between TIRAP and p38 MAPK. Further, manual in-silico phosphorylations of TIRAP tyrosines; Y86, Y106, Y159, and Y187 was created in the Discovery Studio tool to study the conformational changes in protein docking and their binding affinities with p38 MAPK in comparison to non-phosphorylated state. Our molecular docking and 500 ns of molecular dynamic (MD) simulation study demonstrates that the Y86 phosphorylation (pY86) in TIRAP is crucial in promoting the higher binding affinity (∆Gbind) with p38 MAPK. The conformational changes due to the tyrosine phosphorylation mainly at the Y86 site pull the TIRAP closer to the active site in the kinase domain of p38 MAPK and plays a significant role at the interface site which is reversed in its dephosphorylated state. The heatmap of interactions between the TIRAP and p38 MAPK after the MD simulation shows that the TIRAP pY86 structure makes the highest number of stable hydrogen bonds with p38 MAPK residues. Our findings may further be validated in an in-vitro system and would be crucial for targeting the TIRAP and p38 MAPK interaction for therapeutic purposes against the chronic inflammatory response and associated diseases.

Therapeutic effect of Qinghuayin against chronic atrophic gastritis through the inhibition of toll or interleukin-1 receptor domain-containing adaptor inducing interferon-β signaling pathway.

To examine the efficacy of Qinghuayin (, QHY) in rat chronic atrophic gastritis (CAG) models and explored the molecular mechanism of QHY in treating CAG. In total, 65 Wistar rats were randomly divided into the control (= 10) and CAG groups ( = 55). CAG model rats were further divided into five groups: model ( = 10), vitacoenzyme ( = 10), low-dose QHY ( = 10), medium-dose QHY ( = 10), and high-dose QHY groups ( = 10). We analyzed histopathological changes using hematoxylin and eosin staining and measured interleukin (IL)-6 and IL-8 levels in serum using enzyme-linked immunosorbent assay (ELISA) (Boster Bio, Pleasanton, USA). In addition, gastrin (GAS), pepsinogen I (PGI), and PGII expressions were evaluated using ELISA. The protein and mRNA expression of toll-like receptor 4 (TLR4) and toll or interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF) was detected by Western blotting and quantitative reverse transcription-polymerase chain reaction, respectively. Our results revealed that histopathological changes in CAG model rates could be restored by low-, medium-, and high-dose QHY. The changes in GAS and PGI/II expression demonstrated that QHY improved CAG. Serum IL-6 and IL-levels were decreased by QHY administration. TLR4 and TRIF were upregulated at the mRNA and protein levels in the model group but downregulated by QHY administration. We concluded that QHY could effectively improve the histopathological changes of the gastric mucosa induced by CAG in rats. The therapeutic mechanism of QHY may be related to inhibition of the inflammatory factors IL-6 and IL-8 and suppression of TLR4/TRIF mRNA and protein expression.

MiR-3150a-3p, miR-6883-3p and miR-627-5p participate in the phycocyanin-mediated growth diminishment of A549 cells, via regulating a common target toll/interleukin 1 receptor domain-containing adaptor protein

• Phycocyanin diminishes the viability of A549 cells. • Phycocyanin significantly promotes expressions of miR-3150a-3p, miR-6883-3p and miR-627-5p. • TIRAP is a shared directed target of miR-3150a-3p, miR-6883-3p and miR-627-5p. • Knockdown TIRAP expression resulted in similar cell phenotype as miRNAs overexpression. • Overexpression TIRAP restores the diminished A549 viability caused by miRNAs and phycocyanin. As a type of functional food additives, phycocyanin is well-known for its extraordinary antineoplastic activity in non-small cell lung cancer (NSCLC). Previously, a high-throughput miRNA sequencing was employed to excavate miRNAs altered by phycocyanin in A549 cells, while the in-depth regulatory mechanism was inadequate. In this work, three significant up-regulated miRNAs in phycocyanin-treated cells are identified for the first time, including miR-3150a-3p, miR-6883-3p and miR-627-5p. Overexpression of miRNAs obviously diminished the cell growth and colony formation capacity, respectively. Dual-luciferase reporter assay verified that toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) was a direct shared target of three miRNAs. Knockdown the expression of TIRAP resulted in similar cell growth inhibition phenotype as miRNAs overexpression. Strikingly, overexpression of TIRAP could significantly restore the diminished cell viability caused by miRNAs up-regulation, as well as phycocyanin treatment. Consequently, this study proposes that phycocyanin plays an inhibitory effect of NSCLC cells via a miR-3150a-3p/miR-6883-3p/miR-627-5p-TIRAP axis.

Identification of novel inhibitors targeting TIRAP interactions with BTK and PKCδ in inflammation through an in silico approach

ABSTRACT Advanced computational tools focusing on protein–protein interaction (PPI) based drug development is a powerful platform to accelerate the therapeutic development of small lead molecules and repurposed drugs. Toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP) and its interactions with other proteins in macrophages signalling are crucial components of severe or persistent inflammation. TIRAP activation through Bruton’s tyrosine kinase (BTK) and Protein Kinase C delta (PKCδ) is essential for downstream inflammatory signalling. We created homology-based structural models of BTK and PKCδ in MODELLER 9.24. TIRAP interactions with BTK and PKCδ in its non-phosphorylated and phosphorylated states were determined by multiple docking tools including HADDOCK 2.4, pyDockWEB and ClusPro 2.0. Food and Drug Administration (FDA)-approved drugs were virtually screened through Discovery Studio LibDock and Autodock Vina tools to target the common TIR domain residues of TIRAP, which interact with both BTK and PKC at the identified interfacial sites of the complexes. Four FDA-approved drugs were identified and found to have stable interactions over a range of 100 ns MD simulation timescales. These drugs block the interactions of both kinases with TIRAP in silico. Hence, these drugs have the potential to dampen downstream inflammatory signalling and inflammation-mediated disease.

Herbal extracts that induce type I interferons through Toll-like receptor 4 signaling.

BackgroundA mixture of five herbal extracts called internatural (INT), which is prepared from pumpkin seeds, purple turmeric, pearl barley, corn pistil, and cinnamon, is widely used by people in Japan and elsewhere for its immunity-enhancing effects and general health. Although anecdotal evidence indicates its efficacy, the mechanisms by which INT boosts immunity have remained unknown.ObjectiveThe aim of this study was to investigate whether INT induces type I interferons (IFNs) in murine bone marrow-derived macrophages (BMDMs) and by what mechanism.DesignWe measured induction of type I IFNs (IFNβ and IFNα) in BMDMs treated with INT or other Toll-like receptor ligands: bacterial lipopolysaccharides (LPS), dsRNA, poly(I:C), and CpG oligonucleotides. To investigate whether INT signals through Toll-like receptor 4 (TLR4), we tested TLR4-specific inhibitor. We also tested if INT utilizes TLR4 adaptors, toll/IL-1 receptor (TIR) domain-containing adaptor (TRIF), or myeloid differentiation factor 88 (MyD88), we examined INT induction of IFNβ in TRIF-KO and MyD88-KO BMDMs. We then investigated whether INT provides an antiviral effect upon fibroblasts either directly or indirectly using the encephalomyocarditis virus (EMCV) model.ResultsWe first observed that INT, when added to BMDMs, potently induces type I IFNs (IFNβ and IFNα) within 2 h. INT induction of IFN expression was mediated by TLR4, which signaled through the TRIF/MyD88 adaptors, similar to LPS. A high-molecular-weight fraction (MW > 10,000) of INT extracts contained IFN-inducing activity. Supernatants from INT-treated BMDMs protected untreated fibroblast from EMCV infection as reduced viral titers.ConclusionsINT induced type I IFN mRNA and proteins in BMDMs and other cell types. This induction was mediated by TLR4, which transduces signals using the TRIF/MyD88 pathway. The high-MW component of INT contained type I IFN inducing activity. The supernatants from INT-treated cells displayed antiviral activity and protected cells from EMCV infection. These findings indicate that INT is a novel natural IFN inducer that strengthens host’s innate immunity.

DOP52 Development of a host-microbe interaction workflow to reveal the cell- and condition-specific effects of a commensal bacteria upon IBD

Abstract Background Humans are colonized by complex microbial communities which contribute to physiological processes in the host. The communication between microbes and host is crucial to maintain the homeostasis and gut health. Disruption in the microbiome composition leads to increased inflammation and appearance of diseases, such as inflammatory bowel disease (IBD). Interspecies interaction prediction combined with gene expression patterns on individual cell level by single-cell omics data reveals a new insight into the molecular background of cell-type specific host-microbe interactions. Methods Previously we developed the MicrobioLink pipeline (Andrighetti et al, Cells, 2020), an in silico microbe-host protein-protein interaction prediction algorithm. Here, we implemented a computational workflow based on MicrobioLink to predict and compare the cell-specific effects of a commensal bacteria in healthy and diseased conditions using a publicly available single-cell RNAseq dataset (Smilie et al, Cell, 2019) from colon biopsies describing 51 cell types - including fibroblasts, epithelial and immune cells - in healthy, non-inflamed and inflamed ulcerative colitis (UC). With functional analysis, microbe-affected processes have been discovered, while reliable network biology resources, such as OmniPath and Reactome, were used to identify the direct mechanism of action of the bacterial molecules. Results Demonstrating the applicability of the new computational workflow, we analysed the effect of a common gut commensal bacteria - Bacteroides thetaiotaomicron (Bt) - on human immune cells focusing on the Toll-like receptor (TLR) signalling. We found that extracellular vesicles (EVs) secreted by Bt may be able to modulate the TLR pathway intracellularly. The analysis highlighted that Bt targets differ among cells and between the same cells in healthy versus UC conditions. The in silico findings were validated in EV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in EV-elicited NF-kB activation. Conclusion The current pipeline offers potentially interesting connection points and detailed mechanistic insight containing mechanistic information about microbe-host interactions. This information can be tested and harnessed to understand better how microbial proteins may be of therapeutic value in inflammatory diseases, such as IBD.

TIRAP in the Mechanism of Inflammation.

The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) represents a key intracellular signalling molecule regulating diverse immune responses. Its capacity to function as an adaptor molecule has been widely investigated in relation to Toll-like Receptor (TLR)-mediated innate immune signalling. Since the discovery of TIRAP in 2001, initial studies were mainly focused on its role as an adaptor protein that couples Myeloid differentiation factor 88 (MyD88) with TLRs, to activate MyD88-dependent TLRs signalling. Subsequent studies delineated TIRAP’s role as a transducer of signalling events through its interaction with non-TLR signalling mediators. Indeed, the ability of TIRAP to interact with an array of intracellular signalling mediators suggests its central role in various immune responses. Therefore, continued studies that elucidate the molecular basis of various TIRAP-protein interactions and how they affect the signalling magnitude, should provide key information on the inflammatory disease mechanisms. This review summarizes the TIRAP recruitment to activated receptors and discusses the mechanism of interactions in relation to the signalling that precede acute and chronic inflammatory diseases. Furthermore, we highlighted the significance of TIRAP-TIR domain containing binding sites for several intracellular inflammatory signalling molecules. Collectively, we discuss the importance of the TIR domain in TIRAP as a key interface involved in protein interactions which could hence serve as a therapeutic target to dampen the extent of acute and chronic inflammatory conditions.