o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Longifolioside A inhibits TLR4-mediated inflammatory responses by blocking PKCδ activation in LPS-stimulated THP-1 macrophages.

Longifolioside A is an iridoid glucoside compound isolated from Pseudolysimachion rotundum var. subintegrum, which has been used in traditional herbal medicines to treat respiratory inflammatory diseases. Logifolioside A is a potent antioxidant; however, its underlying pharmacological mechanisms of action in inflammatory diseases are unknown. Here, we investigated the inhibitory effects of longifolioside A in lipopolysaccharide (LPS)-stimulated toll-like receptor 4 (TLR4) signal transduction systems using human THP-1 macrophages and HEK293 cells stably expressing human TLR4 protein (293/HA-hTLR4). Longifolioside A significantly reduced the release of inflammatory cytokines such as interleukin (IL)-6, -8, and tumor necrosis factor (TNF)-α in LPS-stimulated THP-1 macrophages. Furthermore, longifolioside A inhibited the expression of inflammatory mediator genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 that produce nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. Longifolioside A suppressed the phosphorylation of PKCδ, IRAK4, IKKα/ß, IκBα, and mitogen-activated protein (MAP) kinases (ERK 1/2 and JNK, but not p38), thereby inactivating the nuclear localization of NF-κB and AP-1, and thus decreasing the expression of inflammatory response genes. Notably, longifolioside A disrupted the interaction between human TLR4 and the TIR domain-containing adaptor protein (TIRAP), an early step during TLR4 activation, thereby reducing IL-8 secretion in 293/HA-hTLR4 cells. This inhibitory effect was comparable to that of TAK-242 (a TLR4 inhibitor, or resatorvid). Our results indicate that longifolioside A prevents inflammatory response by suppressing TLR4 activation required for NF-κB and AP-1 activation.

Aging and contribution of MyD88 and TRIF to expression of TLR pathway-associated genes following stimulation with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease, including socio-economic status, genetics and age; however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft- and hard-tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in the host response to P. gingivalis. The focus of the current study was to use microarray analysis to define the contributions of the TLR adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), and aging, on the expression of TLR pathway-associated mRNAs in response to P. gingivalis. MATERIAL AND METHODS: Bone marrow-derived macrophages (BMØ) from wild-type (Wt), MyD88 knockout (MyD88-KO) and Trif(Lps2) [i.e. containing a point mutation in the lipopolysaccharide 2 (Lps2) gene rendering the Toll/interleukin (IL)-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) protein nonfunctional] mice, at 2-and 12-mo of age, were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison with expression of genes in BMØ cultured in medium only. RESULTS: Using, as criteria, a twofold increase or decrease in mRNA expression, differential expression of 32 genes was observed when Wt BMØ from 2-mo-old mice were cultured with P. gingivalis compared with the medium-only control. When compared with 2-mo-old Wt mice, 21 and 12 genes were differentially expressed (p < 0.05) as a result of the mutations in MyD88 or TRIF, respectively. The expression of five genes was significantly (p < 0.05) reduced in Wt BMØ from 12-mo-old mice compared with those from 2-mo-old mice following culture with P. gingivalis. Age also influenced the expression of genes in MyD88-KO and Trif(Lps2) mice challenged with P. gingivalis. CONCLUSIONS: Our results indicate that P. gingivalis induces differential expression of TLR pathway-associated genes, and both MyD88 and TRIF play roles in the expression of these genes. Age also played a role in the expression of TLR-associated genes following stimulation of BMØ with P. gingivalis.

Characterization of the interleukin 1 receptor-associated kinase 4 (IRAK4)-encoding gene in salmonid fish: the functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells.

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells.

Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

Serum SARM1 Levels and Diabetic Peripheral Neuropathy in Type 2 Diabetes: Correlation with Clinical Neuropathy Scales and Nerve Conduction Studies and Impact of COVID-19 vaccination.

Patients with peripheral neuropathy with type 2 diabetes mellitus (T2DM) are more likely to have functional impairments. Recently, the gene for serum sterile alpha and toll/interleukin receptor motif-containing protein 1 (SARM1), which may contribute to the pathogenesis of Wallerian degeneration, was discovered in mice models of peripheral neuropathy. We set out to assess serum SARM1's activity as a potential biomarker for the early identification of diabetic peripheral neuropathy in T2DM patients while also examining the impact of the COVID-19 vaccine on SARM1 levels. We assessed the cross-sectional relationships between the SARM1 biomarker, clinical neuropathy scales, and nerve conduction parameters in 80 participants aged between 30 years and 60 years. The analysis was carried out after the patients were split into two groups since we discovered a significant increase in SARM1 levels following the second dose of the COVID-19 vaccination, where group A received one dose of the COVID-19 vaccine inoculation, and group B received two doses of the COVID-19 vaccine. SARM1 was correlated significantly (p < 0.05) with MNSIe and NSS in group A and showed a consistent positive correlation with the other neuropathy clinical scales in group A and group B without reaching statistical significance. Additionally, SARM1 was negatively correlated significantly (p < 0.05) with the median sensory amplitude in group A and showed a consistent negative correlation with the six other sensory and motor nerves' potential amplitude in group A and group B without reaching statistical significance. In conclusion, SARM1 showed a consistent correlation with clinical neuropathy scales and nerve conduction parameters after accounting for the influence of COVID-19 vaccination doses.

Crystallization and preliminary X-ray diffraction analyses of the TIR domains of three TIR-NB-LRR proteins that are involved in disease resistance in Arabidopsis thaliana.

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)-leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 Šresolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 Šresolution, had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 Šresolution and had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity.

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Background: Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. Methods: The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. Results: 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. Conclusion: The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

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

OBJECTIVE: 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-ß (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

EDS1 modules as two-tiered receptor complexes for TIR-catalyzed signaling molecules to activate plant immunity.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain detect pathogen effectors to produce TIR-catalyzed signaling molecules for activation of plant immunity. Plant immune signaling by TIR-containing NLR (TNL) proteins converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TNL signaling also require helper NLRs N requirement gene 1 (NRG1) and activated disease resistance 1 (ADR1). In two recent remarkable papers published in Science, the authors show that the TIR-containing proteins catalyze and produce two types of signaling molecules, ADPr-ATP/diADPR and pRib-AMP/ADP. Importantly, they demonstrate that EDS1-SAG101 and EDS1-PAD4 modules are the receptor complexes for ADPr-ATP/diADPRp and Rib-AMP/ADP, respectively, which allosterically promote EDS1-SAG101 interaction with NRG1 and EDS1-PAD4 interaction with ADR1. Thus, two different small molecules catalyzed by TIR-containing proteins selectively activate the downstream two distinct branches of EDS1-mediated immune signalings. These breakthrough studies significantly advance our understanding of TNL downstream signaling pathway.

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy.

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.

In silico structural homology modeling and characterization of multiple N-terminal domains of selected bacterial Tcps.

Several bacterial pathogens produce Toll/interleukin-1 receptor (TIR) domain-containing protein homologs that are important for subverting the Toll-like receptor (TLR) signaling cascades in hosts. Consequently, promoting the persistence and survival of the bacterial pathogens. However, the exact molecular mechanisms elucidating the functional characteristics of these bacterial proteins are not clear. Physicochemical and homology modeling characterization studies have been conducted to predict the conditions suitable for the stability and purification of these proteins and to predict their structural properties. The outcomes of these studies have provided important preliminary data for the drug discovery pipeline projects. Here, using in silico physicochemical and homology modeling tools, we have reported the primary, secondary and tertiary structural characteristics of multiple N-terminal domains of selected bacterial TIR domain-containing proteins (Tcps). The results show variations between the primary amino acid sequences, secondary structural components and three-dimensional models of the proteins, suggesting the role of different molecular mechanisms in the functioning of these proteins in subverting the host immune system. This study could form the basis of future experimental studies advancing our understanding of the molecular basis of the inhibition of the host immune response by the bacterial Tcps.

Discovery of a Family of Mixed Lineage Kinase Domain-like Proteins in Plants and Their Role in Innate Immune Signaling.

HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.

TIR Domain Proteins Are an Ancient Family of NAD+-Consuming Enzymes.

The Toll/interleukin-1 receptor (TIR) domain is the signature signaling domain of Toll-like receptors (TLRs) and their adaptors, serving as a scaffold for the assembly of protein complexes for innate immune signaling [1, 2]. TIR domain proteins are also expressed in plants, where they mediate disease resistance [3, 4], and in bacteria, where they have been associated with virulence [5-9]. In pursuing our work on axon degeneration [10], we made the surprising discovery that the TIR domain of SARM1 (sterile alpha and TIR motif containing 1), a TLR adaptor protein, has enzymatic activity [11]. Upon axon injury, the SARM1 TIR domain cleaves nicotinamide adenine dinucleotide (NAD+), destroying this essential metabolic co-factor to trigger axon destruction [11, 12]. Whereas current studies of TIR domains focus on their scaffolding function, our findings with SARM1 inspired us to ask whether this enzymatic activity is the primordial function of the TIR domain. Here we show that ancestral prokaryotic TIR domains constitute a new family of NADase enzymes. Using purified proteins from a cell-free translation system, we find that TIR domain proteins from both bacteria and archaea cleave NAD+ into nicotinamide and ADP-ribose (ADPR), with catalytic cleavage executed by a conserved glutamic acid. A subset of bacterial and archaeal TIR domains generates a non-canonical variant cyclic ADPR (cADPR) molecule, and the full-length TIR domain protein from pathogenic Staphylococcus aureus induces NAD+ loss in mammalian cells. These findings suggest that the primordial function of the TIR domain is the enzymatic cleavage of NAD+ and establish TIR domain proteins as a new class of metabolic regulatory enzymes.

Toll-like receptor signaling and serum levels of interferon ß and lipopolysaccharide binding protein are related to abdominal obesity: a case-control study between metabolically healthy and metabolically unhealthy obese individuals.

It is still unclear whether toll-like receptor (TLR) signaling and serum levels of inflammatory markers in metabolically unhealthy abdominally obese (MUAO) are due to their obesity and/or their metabolic state. We hypothesized that abdominal obesity is an important mediator of the association of metabolic state with TLR signaling and serum inflammatory markers. Therefore, in this case-control study, we compared the expression levels of TLR4 and Toll/interleukin-1 receptor domain containing adaptor protein-inducing interferon ß (TRIF) and serum concentrations of interferon ß and lipoprotein-binding protein (LBP) in metabolically healthy abdominally obese (MHAO) and MUAO individuals. Basal blood samples from 65 abdominally obese subjects with waist circumference (WC) of at least 95 cm were collected to determine serum metabolic parameters, IFNß, and LBP. Those with 3 or more metabolic alterations were defined as MUAO (n = 34), and those having 2 or less were classified as MHAO (n = 31). Furthermore, messenger RNA (mRNA) was isolated from peripheral blood mononuclear cells. TLR4 and TRIF gene expression assay was performed using quantitative real-time polymerase chain reaction. There were significant differences in serum fasting blood sugar (P = .017), triglyceride (P < .001), cholesterol (P = .002), and low-density lipoprotein cholesterol (P = .034) between the MUAO and MHAO groups, whereas no significant difference was observed in the expression ratio of TLR4 and TRIF mRNA and serum levels of IFNß and LBP. However, a significant correlation was noticed between mRNA expression levels of TLR4 and TRIF (r = 0.50, P < .001) and serum IFNß and LBP (r = 0.70, P < .001). It is concluded that the expression levels of TLR4 and TRIF as well as serum IFNß and LBP are more related to abdominal obesity than to metabolic health.

Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein.

The toll/interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) plays an important role in the toll-like receptor (TLR) 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) on the plasma membrane and PI (3,4,5)-trisphosphate (PIP3) on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP's membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein-protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD) for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM) contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32), additional positively charged residues (K34, K35, and R36) showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP's affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond) and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP's membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.

The SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD+ Cleavage Activity that Promotes Pathological Axonal Degeneration.

Axonal degeneration is an early and prominent feature of many neurological disorders. SARM1 is the central executioner of the axonal degeneration pathway that culminates in depletion of axonal NAD+, yet the identity of the underlying NAD+-depleting enzyme(s) is unknown. Here, in a series of experiments using purified proteins from mammalian cells, bacteria, and a cell-free protein translation system, we show that the SARM1-TIR domain itself has intrinsic NADase activity-cleaving NAD+ into ADP-ribose (ADPR), cyclic ADPR, and nicotinamide, with nicotinamide serving as a feedback inhibitor of the enzyme. Using traumatic and vincristine-induced injury models in neurons, we demonstrate that the NADase activity of full-length SARM1 is required in axons to promote axonal NAD+ depletion and axonal degeneration after injury. Hence, the SARM1 enzyme represents a novel therapeutic target for axonopathies. Moreover, the widely utilized TIR domain is a protein motif that can possess enzymatic activity.

Bioinformatic Analysis of Toll-Like Receptor Sequences and Structures.

Continual advancements in computing power and sophistication, coupled with rapid increases in protein sequence and structural information, have made bioinformatic tools an invaluable resource for the molecular and structural biologist. With the degree of sequence information continuing to expand at an almost exponential rate, it is essential that scientists today have a basic understanding of how to utilise, manipulate and analyse this information for the benefit of their own experiments. In the context of Toll-Interleukin I Receptor domain containing proteins, we describe here a series of the more common and user-friendly bioinformatic tools available as Internet-based resources. These will enable the identification and alignment of protein sequences; the identification of functional motifs; the characterisation of protein secondary structure; the identification of protein structural folds and distantly homologous proteins; and the validation of the structural geometry of modelled protein structures.

S100A proteins as molecular targets in the ocular surface inflammatory diseases.

The S100 proteins are calcium-binding proteins that are exclusively expressed in vertebrates, where they interact with enzymes, cytoskeletal proteins, receptors, transcription factors, and nucleic acids to regulate proliferation, differentiation, apoptosis, inflammation, cell migration, energy metabolism, and Ca(2+) homeostasis. In this review, we focus on the S100A8 and S100A9 members of the family that are involved in the regulation of neutrophil chemotaxis and inflammation related to ocular surface diseases such as dry eye, meibomian gland dysfunction, pterygium, and corneal neovascularization. In our previous studies, we have found that the levels of S100A8 and S100A9 were elevated in these inflammatory ocular diseases. For instance, S100A8 and A9 were found to be upregulated in pterygium tissues at both transcript and protein levels. These findings are consistent with the role of S100A8 and S100A9 proteins in activating the innate immune system in the eye via Toll-like receptors (TLRs) and altering the immune tolerance of the eye-associated lymphoid system. Recently, use of S100A8-targeting antibody has shown promising results in targeting corneal neovascularization. Injection of S100A8 has been shown to inhibit eosinophilic infiltration and thus may have potential therapeutic implications in allergic diseases.

Research on the association of TIRAP coding region polymorphism with susceptibility to tuberculosis in Chinese Han population / 中华微生物学和免疫学杂志

Objective To detect specific polymorphisms in Toll-interleukin 1 receptor domain containing adaptor protein(TIRAP) coding region for Chinese Han population, and verify whether they are associated with susceptibility to tuberculosis. Methods Search TIRAP polymorphisms by sequencing in small sample; detect single nucleotide polymorphism(SNP) by ligase detection reaction technique in large sample; analyze whether polymorphisms are related to tuberculosis by statistic methods. Results Four polymorphisms were present in the TIRAP coding region. 394A had higher frequencies in the tuberculosis(TB)group than the control. But allelic and genotypic analysis showed that there were no significant difference in statistic between TB patients and controls(P>0.05). The SNP G164A mutation related with TB patient's condition. Comparing to controls, retreatment patients' allelic frequencies had significant difference in statistic(P<0.05), sputum positive patients and lung cavitation patients had lower 164A frequencies. Conclusion TIRAP coding region polymorphisms may be risk factors for TB occurrence and development in Chinese Han population.