Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG-labeled derivative of the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (Pam(3)CSK(4)) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam(3)CSK(4)-FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam(3)CSK(4)-FLAG binding, CD14 and Pam(3)CSK(4)-FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low-mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.
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PMID:Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1. 1571 90

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)). However, examination of a wide variety of lipopeptide derivatives indicates that recognition by human TLR1 and -2 does not strictly correlate with the number or position of the acyl chains on the modified cysteine residue. Conversely, human TLR2 and -6 exclusively respond to lipopeptides possessing a diacylglycerol group. Most surprisingly, we have found that an R stereoisomer of diacylated macrophage-activating lipopeptide 2 (MALP-2) exclusively activates epithelial cells through TLR6 and -2 but not through TLR1 and -2. These results suggest that the chirality of the central carbon of the diacylglycerol group of these agonists is a structural determinant for human TLR recognition. Examination of chimeric receptors, generated by domain exchange between TLR1 and -6, has revealed that leucine-rich repeats 9-12 of the extracellular domain enable these receptors to discriminate between structurally similar lipopeptides. However, additional chimeric constructs reveal that this region alone is not sufficient to generate receptors that can functionally cooperate with TLR2. Our results support the idea that TLR1 and TLR6 diverged during evolution to differentially recognize natural lipoprotein structures and that this function has been conserved with respect to the human receptors.
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PMID:Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination. 1612 84

It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-kappaB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-kappaB luciferase reporter gene, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam(3)CSK(4)) and Pam(3)CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14(dS39-A48), a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2(dE772-S784)) or TLR1 (TLR1(dQ636-K779)) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam(3)CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam(3)CSK(4) at a dissociation constant of 5.7 microM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.
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PMID:CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex. 1684 91

The Toll/Interleukin-1 receptor (TIR) domain of the Toll-like receptors (TLRs) plays an important role in innate host defense signaling. The TIR-TIR platform formed by the dimerization of two TLRs promotes homotypic protein-protein interactions with additional cytoplasmic adapter molecules to form an active signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes. To generate a better understanding of the functional domains of TLR2 we performed a random mutagenesis analysis of the human TLR2 TIR domain and screened for TLR2/1 signaling-deficient mutants. Based upon the random mutagenesis results, we performed an alanine scanning mutagenesis of the TLR2 DD loop and part of the alphaD region. This resulted in the identification of four residues crucial for TLR2/1 signaling: Arg-748, Phe-749, Leu-752, and Arg-753. Computer-assisted energy minimization and docking studies indicated three regions of interaction in the TLR2/1 TIR-docked heterodimer. In Region I, residues Arg-748 and Phe-749 in TLR2 DD loop were involved in close contacts with Gly-676 in the TLR1 BB loop. Because this model suggested that steric hindrance would significantly alter the binding interactions between DD loop of TLR2 and BB loop of TLR1, Gly-676 in TLR1 was rationally mutated to Ala and Leu. As expected, in vitro functional studies involving TLR1 G676A and TLR1 G676L resulted in reduced PAM(3)CSK(4) mediated NF-kappaB activation lending support to the computerized predictions. Additionally, mutation of an amino acid residue (TLR2 Asp-730) in Region II also resulted in decreased activity in agreement with our model, providing new insights into the structure-function relationship of TLR2/1 TIR domains.
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PMID:Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling. 1689 94

Bacteremia is one of the leading causes of death by infectious disease. To understand the immune mechanisms required for the rapid control of bacteremia, we studied Borrelia hermsii, a bacterial pathogen that colonizes the blood stream of humans and rodents to an extremely high density. A T cell-independent IgM response is essential and sufficient for controlling B. hermsii bacteremia. Mice deficient in Bruton's tyrosine kinase (Btk), despite their known defect in BCR signaling, generated B. hermsii-specific IgM and resolved bacteremia, suggesting that an alternative activation or costimulatory pathway remained functional for T cell-independent B cells in Btk(-/-) mice. B. hermsii contains putative ligands for TLRs, and we found that mice deficient in TLR1, TLR2, or the TLR adaptor MyD88 generated anti-B. hermsii IgM with delayed kinetics and suffered more severe episodes of bacteremia. In striking contrast to the anti-B. hermsii IgM response in mice deficient only in Btk, mice deficient in both Btk and MyD88 were entirely incapable of generating B. hermsii-specific Ab or resolving bacteremia. The response to a T cell-dependent model Ag was unaffected in Btk(-/-) x MyD88(-/-) mice. These results suggest that MyD88 specifically promotes T cell-independent BCR signaling and that, in the absence of Btk, this TLR-mediated stimulation is a required component of this signal.
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PMID:MyD88- and Bruton's tyrosine kinase-mediated signals are essential for T cell-independent pathogen-specific IgM responses. 1733 72

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.
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PMID:The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2. 1751 60

The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.
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PMID:c-Jun N-terminal kinase 1 is required for Toll-like receptor 1 gene expression in macrophages. 1766 70

TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.
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PMID:Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. 1788 40

TAK-242, a small-molecule antisepsis agent, has shown to suppress lipopolysaccharide (LPS)-induced inflammation. In this study, we demonstrate that TAK-242 is a selective inhibitor of Toll-like receptor (TLR)-4 signaling. TAK-242 almost completely suppressed production of nitric oxide (NO) or tumor necrosis factor (TNF)-alpha induced by a TLR4-specific ligand, ultra-pure LPS, in mouse RAW264.7, human U-937 and P31/FUJ cells, whereas this agent showed little effect on other TLR ligands, Pam(3)CSK(4) (TLR1/2), peptidoglycan (TLR2/6), double strand RNA (TLR3), R-848 (TLR7) and CpG oligonucleotide (TLR9). Furthermore, TAK-242 potently inhibited nuclear factor (NF)-kappaB activation induced by ultra-pure LPS in HEK293 cells transiently expressing TLR4 and co-receptors, myeloid differentiation protein-2 (MD2) and CD14, whereas this agent showed little effect on other TLRs, TLR1/2, TLR2/6, TLR3, TLR5, TLR7 and TLR9. TAK-242 also inhibited ligand-independent NF-kappaB activation resulting from over-expression of TLR4. Although chimera receptors, which are consist of the extracellular domain of CD4 and the intracellular domain of human or mouse TLR4, showed constitutive NF-kappaB activation, TAK-242 potently inhibited the signaling from CD4-TLR4 chimera receptors. In contrast, the NF-kappaB activation mediated by TLR4 adaptors, myeloid differentiation factor 88 (MyD88), TIR-associated protein (TIRAP), Toll/IL-1R homology (TIR)-domain-containing adaptor protein-inducing interferon-beta (TRIF) or TRIF-related adaptor molecule (TRAM) was not affected by TAK-242. TAK-242 is therefore a selective inhibitor of signaling from the intracellular domain of TLR4 and represents a novel therapeutic approach to the treatment of TLR4-mediated diseases.
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PMID:TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain. 1829 27

TLR have emerged as important primary sensors for diverse stimuli and are increasingly implicated in various diseases. However, the molecular mechanisms underlying the regulation of the TLR system remain poorly understood. In this study, we report that some PGs may control TLR-mediated inflammatory events through modulation of TLR2 expression in brain immune cells. We first found that 15-deoxy-Delta12,14-PG J(2) (15d-PGJ(2)) markedly altered the expression of TLR2 but not TLR4, TLR1, and TLR9 at the message and protein levels in activated glia. Down-regulation of TLR2 expression and downstream events of TLR2 activation, including phagocytosis by 15d-PGJ(2), were also observed in cells treated with representative TLR2 ligands such as lipoteichoic acid and Pam(3)CSK(4). We further revealed that certain 15d-PGJ(2)-related PGs such as 15d-PGD(2) and PGD(2) also suppressed the ligand-stimulated increase of TLR2 expression, whereas PGE(2) and arachidonic acids did not. Interestingly, TLR2 expression was down-regulated even when such PGs were added at several hours after stimulator treatment. These findings appear to be independent of peroxisome proliferator-activated receptor gamma and D prostanoid receptors (DPs) because potent synthetic peroxisome proliferator-activated receptor gamma agonists, selective DP1 agonist, or DP2 agonist did not mimic the effects of such PGs on TLR2 expression. Taken together, our results suggest that 15d-PGJ(2), 15d-PGD(2), and PGD(2) may play notable roles as modulators of the TLR2-mediated inflammatory events, and provide new insight into the resolution of inflammation in the brain.
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PMID:Regulation of TLR2 expression by prostaglandins in brain glia. 1852 8


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