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Inflammation Research, 2018
M1-like inflammatory phenotype of macrophages plays a critical role in tissue damage in chronic inflammatory diseases. Previously, we found that the nitrone spin trap 5,5-dimethyl-1-pirroline Noxide (DMPO) dampens lipopolysaccharide (LPS)-triggered inflammatory priming of RAW264.7 cells. Herein, we tested whether DMPO by itself can induce changes in macrophage transcriptome, and that these effects may prevent LPS-induced activation of macrophages. To test our hypothesis, we performed a transcriptomic and bioinformatics analysis in RAW264.7 cells incubated with or without LPS, in the presence or in the absence of DMPO. Functional data analysis showed 79 differentially expressed genes (DEGs) when comparing DMPO vs Control. We used DAVID databases for identifying enriched gene ontology terms and Ingenuity Pathway Analysis for functional analysis. Our data showed that DMPO vs Control comparison DEGs are related to downregulation immune-system processes among others. Functional analysis indicated that interferon-response factor 7 and toll-like receptor were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses of the DMPO+LPS vs LPS DEGs were consistent with DMPO dampening LPS-induced inflammatory transcriptomic profile in RAW 264.7. These changes were confirmed using Nanostring technology. Taking together our data, surprisingly, indicates that DMPO by itself affects gene expression related to regulation of immune system and that DMPO dampens LPS-triggered inflammatory transcriptomic profile. Our data provide critical data for further studies on the possible use of DMPO as a structural platform for the design of novel mechanism-based anti-inflammatory drugs.
In Silico Approach to Inhibition of Signaling Pathways of Toll-Like Receptors 2 and 4 by ST2L
PLOS One, 2011
Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.
Proceedings of the National Academy of Sciences, 2015
Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C 16 H 15 NO 4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. small molecule inhibitor | BB loop | TLR2 pocket | CADD T oll-like receptors (TLRs) are type I transmembrane receptors that detect conserved "pathogen-associated molecular patterns" from microbes, as well as host-derived "danger-associated molecular patterns" (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4-9).
Biochimica Et Biophysica Acta: Molecular Basis Of Disease, 2019
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The Role of Protein-Protein Interactions in Toll-Like Receptor Function
Progress in Biophysics and Molecular Biology, 2015
As part of the innate immune system, the Toll-like receptors (TLRs) represent key players in the first line of defense against invading foreign pathogens, and are also major targets for therapeutic immunomodulation. TLRs are type I transmembrane proteins composed of an ectodomain responsible for ligand binding, a single-pass transmembrane domain, and a cytoplasmic Toll/Interleukin-1 receptor (TIR) signaling domain. The ectodomains of TLRs are specialized for recognizing a wide variety of pathogen-associated molecular patterns, ranging from lipids and lipopeptides to proteins and nucleic acid fragments. The members of the TLR family are highly conserved and their ectodomains are composed of characteristic, solenoidal leucine-rich repeats (LRRs). Upon ligand binding, these rigid LRR scaffolds dimerize (or reorganize in the case of pre-formed dimers) to bring together their carboxy-terminal transmembrane and TIR domains. The latter are proposed to act as a platform for recruitment of adaptor proteins and formation of higher-order complexes, resulting in propagation of downstream signaling cascades. In this review, we discuss the protein-protein interactions critical for formation and stability of productive, ligand-bound TLR complexes. In particular, we focus on the large body of high-resolution crystallographic data now available for the ectodomains of homo-and heterodimeric TLR complexes, as well as inhibitory TLR-like receptors, and also consider computational approaches that can facilitate our understanding of the ligand-induced conformational changes associated with TLR function. We also briefly consider what is known about the protein-protein interactions involved in both TLR transmembrane domain assembly and TIRmediated signaling complex formation in light of recent structural and biochemical data.
Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling
Proceedings of the National Academy of Sciences, 2009
Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR؊TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.
Molecular Immunology, 2012
Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligandinduced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes.
Similar Structures but Different Roles – An Updated Perspective on TLR Structures
Frontiers in Physiology, 2011
Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved structures in pathogens, trigger innate immune responses, and prime antigen-specific adaptive immunity. Elucidation of crystal structures of TLRs interacting with their ligands such as TLR1-2 with triacylated lipopeptide, TLR2-6 with diacylated lipopeptide, TLR4-MD-2 with LPS, and TLR3 with double-stranded RNA (dsRNA) have enabled an understanding of the initiation of TLR signaling. Agonistic ligands such as LPS, dsRNA, and lipopeptides induce "m" shaped TLR dimers in which C-termini converge at the center. Such central convergence is necessary to bring the two intracellular receptor TIR domains closer together and promote their dimerization, which serves as an essential step in downstream signaling. In this review, we summarize TLR ECD structures that have been reported to date with special emphasis on ligand recognition and activation mechanism.