Structural insights into pharmacophore-assisted in silico identification of protein–protein interaction inhibitors for inhibition of human toll-like receptor 4 – myeloid differentiation factor-2 (hTLR4−MD-2) complex

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS?TLR4?MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein–protein interaction (PPI) in TLR4?MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4?MD-2) and dimerization (MD-2?TLR4*) protein–protein interaction interfaces in TLR4?MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4?MD-2 protein–protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in μM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4?MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Crystal structures of mouse and human RP105/MD-1 complexes reveal unique dimer organization of the toll-like receptor family

The Toll-like receptor (TLR) 4/MD-2 heterodimer senses lipopolysaccharide (LPS). RP105 (radioprotective 105 kDa), a TLR-related molecule, is similar to TLR4 in that the extracellular leucine-rich repeats associate with MD-1, the MD-2-like molecule. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. LPS-bound MD-2 opens the secondary interface with TLR4, leading to dimerization of TLR4/MD-2. MD-1 also has a hydrophobic cavity that accommodates lipid IVa, a precursor of lipid A, suggesting a role for the RP105/MD-1 heterodimer in sensing LPS or related microbial products. Little is known, however, about the structure of the RP105/MD-1 heterodimer or its oligomer. Here, we have determined the crystal structures of mouse and human RP105/MD-1 complexes at 1.9 and 2.8 Å resolutions, respectively. Both mouse and human RP105/MD-1 exhibit dimerization of the 1:1 RP105/MD-1 complex, demonstrating a novel organization. The “m”-shaped 2:2 RP105/MD-1 complex exhibits an inverse arrangement, with N-termini interacting in the middle. Thus, the dimerization interface of RP105/MD-1 is located on the opposite side of the complex, compared to the 2:2 TLR4/MD-2 complex. These results demonstrate that the 2:2 RP105/MD-1 complex is distinct from previously reported TLR dimers, including TLR4/MD-2, TLR1/TLR2, TLR2/TLR6, and TLR3, all of which facilitate homotypic or heterotypic interaction of the C-terminal cytoplasmic signaling domain.     

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.     

A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4

MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2.LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2.LPS complex. Finally we show that the MD-2.LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2.LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4(+)/MD-2(-) cells in the inflammatory microenvironment.     

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling.     

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C₁₇H₂₇NO_9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.     

Inhibition of myeloid differentiation 1 specifically in colon with antisense oligonucleotide exacerbates dextran sodium sulfate-induced colitis

Myeloid differentiation 1 (MD-1), also known as lymphocyte antigen 86 (Ly86), is a soluble protein homologous to MD-2 and forms a complex with radioprotective 105 (RP105). RP105/MD-1 complex negatively regulates toll-like receptor 4 (TLR4) signaling and is involved in several immune disorders. However, the precise role of MD-1 in inflammatory bowel diseases (IBD) remains poorly understood. To further investigate the involvement of MD-1 in IBD, we inhibited MD-1 in colon with antisense oligonucleotide (AS-ODN) and assessed the effect of MD-1 inhibition on dextran sodium sulfate (DSS)-induced colitis. We discovered that MD-1 protein expression was remarkably decreased in both patients with ulcerative colitis and mice with DSS-induced colitis. For the first time, we showed that oral administration of MD-1 AS-ODN to mice significantly suppressed the MD-1 protein levels in colon rather than systemic tissues. Subsequently, we found that MD-1 AS-ODN treated mice were more susceptible to DSS-induced colitis based on loss of body weight, colon length, histological scores, and disease activity index. MD-1 inhibition also significantly enhanced inflammatory cytokines production such as IL-6 and IL-1β in colons. Finally, mice treated with MD-1 AS-ODN exhibited increased messenger RNA levels of TLR4 and MyD88 after DSS exposure and showed enhanced nuclear factor (NF)-κB activation compared with the control. Taken together, specifically suppression of MD-1 in colon tissues with AS-ODN exacerbates DSS-induced experimental colitis in mice, which is possibly related to activation of TLR4/NF-κB signaling.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.     

Isolation of monomeric and dimeric secreted MD-2. Endotoxin.sCD14 and Toll-like receptor 4 ectodomain selectively react with the monomeric form of secreted MD-2

Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E.MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E.sCD14 to form monomeric E.MD-2 and activate HEK293/TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E.MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E.CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.     

Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.     

Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-induced inflammatory cell responses by altering LPS binding to its receptors

Pulmonary surfactant protein D (SP-D) is a member of the collectin family that plays an important role in regulating innate immunity of the lung. We examined the mechanisms by which SP-D modulates lipopolysaccharide (LPS)-elicited inflammatory cell responses. SP-D bound to a complex of recombinant soluble forms of Toll-like receptor 4 (TLR4) and MD-2 with high affinity and down-regulated tumor necrosis factor-alpha secretion and NF-kappaB activation elicited by rough and smooth LPS, in alveolar macrophages and TLR4/MD-2-transfected HEK293 cells. Cell surface binding of both serotypes of LPS to TLR4/MD-2-expressing cells was attenuated by SP-D. In addition, SP-D significantly reduced MD-2 binding to both serotypes of LPS. A chimera containing the N-terminal region and the collagenous domain of surfactant protein A, and the coiled-coil neck and lectin domains of SP-D, was a weak inhibitor of LPS-induced cell responses and MD-2 binding to LPS, compared with native SP-D. The collagenase-resistant fragment consisting of the neck plus the carbohydrate recognition domain of SP-D also was a very weak inhibitor of LPS activation. This study demonstrates that SP-D down-regulates LPS-elicited inflammatory responses by altering LPS binding to its receptors and reveals the importance of the correct oligomeric structure of the protein in this process.     

Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells

Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation, known as endotoxin tolerance. Induction of LPS tolerance has been reported to correlate with decreased cell surface expression of the LPS receptor complex, Toll-like receptor 4 (TLR4)/MD-2. However, other results have underscored the existence of mechanisms of LPS tolerance that operate downstream of TLR4/MD-2. In the present study we sought to delineate further the molecular basis of LPS tolerance by examining the TLR4 signaling pathway in endotoxin-tolerant cells. Pretreatment of human monocytes with LPS decreased LPS-mediated NF-kappaB activation, p38 mitogen-activated protein kinase phosphorylation, and TNF-alpha gene expression, documenting the induction of endotoxin tolerance. FACS and Western blot analyses of LPS-tolerant monocytes showed increased TLR2 expression, whereas TLR4 expression levels were not affected. Comparable levels of mRNA and protein for myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNFR-associated factor-6 were found in normal and LPS-tolerant monocytes, while MD-2 mRNA expression was slightly increased in LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1 was not activated in response to LPS stimulation. Moreover, endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also showed decreased IRAK-1 kinase activity in response to LPS despite the failure of LPS to inhibit cell surface expression of transfected TLR4 and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant phenotype.     

Taxanes inhibit human TLR4 signaling by binding to MD-2

LPS is the primary ligand of Toll-like receptor 4, activating it through binding to its accessory protein MD-2. Murine but not human cells expressing MD-2/TLR4 are also activated by paclitaxel. Paclitaxel binds to human MD-2. The binding site of paclitaxel overlaps with the binding site of bis-ANS and LPS, which results in the ability of taxanes to inhibit LPS signaling in the system with human receptors. Circular dichroic spectra of human MD-2 indicated differences in the chemical environment in the presence of paclitaxel and docetaxel. Molecular docking identified the interacting residues of MD-2 and suggests that hydrophobic interactions govern the binding, while the C-3′N group where the paclitaxel and docetaxel differ is exposed on the surface of MD-2.     

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.     

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys¹³³ and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor α production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation.TLR4 is a receptor for lipopolysaccharide (LPS)⁴ a major constituent of outer membrane of Gram-negative bacteria. MD-2 is the final LPS-binding protein in the recognition cascade before TLR4 transmits the signal across the cell membrane to activate the inflammatory response. MD-2 binds to the ectodomain of TLR4 and binds LPS either alone or in complex with TLR4 (1, 2). Mice deficient in MD-2 survive endotoxic shock (3). MD-2 has been indispensable in almost all investigated conditions of TLR4-triggered inflammation; therefore, it could represent the “Achilles'' heel” of the inflammatory response to LPS and a target for a pharmacological intervention in endotoxemia as well as other conditions involving cell activation mediated by TLR4 (4, 5). The existence of a single free cysteine residue among the seven cysteine residues has been predicted from MD-2 mutagenesis (6, 7) and molecular modeling proposed that Cys¹³³ lies in the hydrophobic pocket (8, 9). The hydrophobic binding site of MD-2 was also mapped by an apolar probe, bis-ANS, which does not contain acyl chains as most LPS antagonists yet preserves the characteristic structural motif of lipid A, consisting of a hydrophobic region and a pair of separated negatively charged groups (10). Crystal structures of MD-2 with bound eritoran or lipid IVa confirmed the location of Cys¹³³ in the hydrophobic pocket in close vicinity of bound lipid A derivatives (11, 12). The free cysteine residue inside the binding pocket can thus be a target for irreversible inhibition of MD-2 activity. An inhibitory mechanism based on a covalent modification of a free cysteine residue in the active or binding site of a protein has been demonstrated for other proteins, such as in cysteine proteases, where the cysteine residue participates in the catalytic triad (13), in cholesteryl ester transfer protein (CETP) (14), IκB kinase (15), thioredoxin reductase (16), and sortase (17).In our study, we investigated the possibility of targeting free cysteine residue of MD-2 for the inhibition of LPS signaling. We determined covalent binding into the hydrophobic pocket of MD-2 for fluorescent compounds 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and N-pyrene maleimide. Drugs JTT-705 and auranofin, already in use for alternative indications, were also shown to bind to MD-2 and decrease LPS signaling. The identity of Cys¹³³ as the residue responsible for this interaction was demonstrated by point mutagenesis. Our results confirm that the proposed mechanism of inhibition of MD-2 can have potential therapeutic value but may also have a physiological role.     

A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.     

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.     

The extra domain A of fibronectin activates Toll-like receptor 4

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.