TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123-129) and loop EF (81-89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

Streptococcus pneumoniae is a polysaccharide-encapsulated bacterium. The capsule thickens during blood invasion compared with the thinner capsules observed in asymptomatic nasopharyngeal colonization. However, the underlying mechanism regulating differential CPS expression remains unclear. CPS synthesis requires energy that is supplied by ATP. Previously, we demonstrated a correlation between ATP levels and adenylate kinase in S. pneumoniae (SpAdK). A dose-dependent induction of SpAdK in serum was also reported. To meet medical needs, this study aimed to elucidate the role of SpAdK in the regulation of CPS production. CPS levels in S. pneumoniae type 2 (D39) increased proportionally with SpAdK levels, but they were not related to pneumococcal autolysis. Moreover, increased SpAdK levels resulted in increased total tyrosine kinase Cps2D levels and phosphorylated Cps2D, which is a regulator of CPS synthesis in the D39 strain. Our results also indicated that the SpAdK and Cps2D proteins interact in the presence of Mg-ATP. In addition, in silico analysis uncovered the mechanism behind this protein-protein interaction, suggesting that SpAdK binds with the Cps2D dimer. This established the importance of the ATP-binding domain of Cps2D. Taken together, the biophysical interaction between SpAdK and Cps2D plays an important role in enhancing Cps2D phosphorylation, which results in increased CPS synthesis.

Middle East respiratory syndrome coronavirus (MERS-CoV) has evolved to navigate through the sophisticated network of a host's immune system. The immune evasion mechanism including type 1 interferon and protein kinase R-mediated antiviral stress responses has been recently attributed to the involvement of MERS-CoV protein 4a (p4a) that masks the viral dsRNA. However, the structural mechanism of how p4a recognizes and establishes contacts with dsRNA is not well explained. In this study, we report a dynamic mechanism deployed by p4a to engage the viral dsRNA and make it unavailable to the host immune system. Multiple variants of p4a-dsRNA were created and investigated through extensive molecular dynamics procedures to highlight crucial interfacial residues that may be used as potential pharmacophores for future drug development. The structural analysis revealed that p4a exhibits a typical αβββα fold structure, as found in other dsRNA-binding proteins. The α1 helix and the β1-β2 loop play a crucial role in recognizing and establishing contacts with the minor grooves of dsRNA. Further, mutational and binding free energy analyses suggested that in addition to K63 and K67, two other residues, K27 and W45, might also be crucial for p4a-dsRNA stability.

Gp41 is an envelope glycoprotein of human immune deficiency virus (HIV). HIV viral glycoprotein gp41, present in complex with gp120, assists the viral entry into host cell. Over eighty thousands individuals are HIV infected in Pakistan which makes about 0.2% of 38.6 million infected patients worldwide. Hence, HIV gp41 protein sequences isolated in Pakistan were analyzed for the CD4 and CD8 T cells binding epitopes.

Negative regulation of Toll-like receptor-4 (TLR4) is anticipated to control the pathogen-induced exaggerated immune response. However, effective TLR4 antagonists with scarce off-target effects are yet to be developed. To fill this void, we sought to design small peptide-inhibitors of the TLR4/MD2-LPS interaction. Here we report novel TLR4-antagonistic peptides (TAP), identified through phage display, endowed with the LPS-induced proinflammation inhibition, and confirmed in mice. TAPs-attributed TLR4-antagonism were initially evaluated through NF-κB inhibition in HEK-blue hTLR4 and RAW264.7 cells, and further reinforced by the downregulation of MAPKs (mitogen-activated protein kinases), NF-κB, interleukin 6, and suppression of the oxidative-stress products and iNOS in macrophages and human peripheral blood mononuclear cells (hPBMCs). Among these, TAP2 specifically halted the TLR4, but not other TLRs signaling, which was further confirmed by the biophysical kinetic assay. Finally, TAP2 diminished LPS-elicited systemic cytokine response in vivo, suggesting that TAPs, specifically TAP2, have the potential to treat TLR4-mediated immune ailments.

Despite being crucial for combating microbes, paradoxical Toll-like receptors (TLRs) signaling have been associated with the aggravation of multiple immune disorders such as systemic lupus erythematosus, psoriasis, rheumatoid arthritis, and nonalcoholic steatohepatitis. The stoichiometry and precise arrangement of the interaction of adapters (via their Toll/interleukin-1 receptor [TIR] domains) are indispensable for the activation of TLRs and of downstream signaling cascades. Among adapters, plasma membrane-anchored MyD88 adaptor-like (MAL) has the potential for BB-loop-mediated self-oligomerization and interacts with other TIR domain-containing adaptors through αC and αD helices. Here, we used information on the MAL-αC interface to exploit its pharmacophores and to design a decoy peptide (MIP2) with broad-range TLR-inhibitory abilities. MIP2 abrogated MyD88- and TRIF-dependent lipopolysaccharide (LPS)-induced TLR4 signaling in murine and human cell lines and manifested a therapeutic potential in models of psoriasis, systemic lupus erythematosus, nonalcoholic steatohepatitis, and sepsis. Levels of hallmark serological and histological biomarkers were significantly restored and the disease symptoms were substantially ameliorated by MIP2 treatment of the animals. Collectively, our biophysical, in vitro, and in vivo findings suggest that MIP2 has broad specificity for TLRs and may be effective in modulating autoimmune complications caused by microbial or environmental factors.

Mutations affect gene functions related to cancer behavior, including cell growth, metastasis, and drug responses. Genome-wide profiling of cancer mutations and drug responses has identified actionable targets that can be utilized for the management of cancer patients. Here, the recapitulation of pharmacogenomic data revealed that the mutation of EPHB6 is associated with paclitaxel resistance in cancer cells. Experimental data confirmed that the EPHB6 mutation induces paclitaxel resistance in various cancer types, including lung, skin, and liver cancers. EPHB6 mutation-induced paclitaxel resistance was mediated by an interaction with EPHA2, which promotes c-Jun N-terminal kinase (JNK)-mediated cadherin 11 (CDH11) expression. We demonstrated that EPHB6-mutated cells acquire cell adhesion-mediated drug resistance (CAM-DR) in association with CDH11 expression and RhoA/focal adhesion kinase (FAK) activation. Targeted inhibition of EPHA2 or CDH11 reversed the acquired paclitaxel resistance, suggesting its potential clinical utility. The present results suggest that the EPHB6 mutation and its downstream EPHA2/JNK/CDH11/RhoA/FAK signaling axis are novel diagnostic and therapeutic targets for overcoming paclitaxel resistance in cancer patients.

Direct inhibition of tumor necrosis factor-alpha (TNF-α) action is considered a promising way to prevent or treat TNF-α-associated diseases. The trimeric form of TNF-α binds to its receptor (TNFR) and activates the downstream signaling pathway. The interaction of TNF-α with molecular-grade dimethyl sulfoxide (DMSO) in an equal volumetric ratio renders TNF-α inert, in this state, TNF-α fails to activate TNFR. Here, we aimed to examine the inhibition of TNF-α function by various concentrations of DMSO. Its higher concentration led to stronger attenuation of TNF-α-induced cytokine secretion by fibroblasts, and of their death. We found that this inhibition was mediated by a perturbation in the formation of the functional TNF-α trimer. Molecular dynamics simulations revealed a transient interaction between DMSO molecules and the central hydrophobic cavity of the TNF-α homodimer, indicating that a brief interaction of DMSO with the TNF-α homodimer may disrupt the formation of the functional homotrimer. We also found that the sensitizing effect of actinomycin D on TNF-α-induced cell death depends upon the timing of these treatments and on the cell type. This study will help to select an appropriate concentration of DMSO as a working solvent for the screening of water-insoluble TNF-α inhibitors.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo--a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

Toll-like receptors (TLRs) play a fundamental role in the inflammatory response against invading pathogens. However, the dysregulation of TLR-signaling pathways is implicated in several autoimmune/inflammatory diseases. Here, we show that a novel small molecule TLR-inhibitor (TAC5) and its derivatives TAC5-a, TAC5-c, TAC5-d, and TAC5-e predominantly antagonized poly(I:C) (TLR3)-, imiquimod (TLR7)-, TL8-506 (TLR8)-, and CpG-oligodeoxynucleotide (TLR9)-induced signaling pathways. TAC5 and TAC5-a significantly hindered the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), reduced the phosphorylation of mitogen-activated protein kinases, and inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6. Besides, TAC5-a prevented the progression of psoriasis and systemic lupus erythematosus (SLE) in mice. Interestingly, TAC5 and TAC5-a did not affect Pam3CSK4 (TLR1/2)-, FSL-1 (TLR2/6)-, or lipopolysaccharide (TLR4)-induced TNF-α secretion, indicating their specificity towards endosomal TLRs (TLR3/7/8/9). Collectively, our data suggest that the TAC5 series of compounds are potential candidates for treating autoimmune diseases such as psoriasis or SLE.

Inflammatory bowel disease (IBD) is a highly prevalent gut inflammatory disorder. Complicated clinical outcomes prolong the use of conventional therapy and often lead to compromised immunity followed by adverse events and high relapse rates. Thus, a profound medical intervention is required. Previously, intranasal immunization of pneumococcal pep27 mutant (Δpep27) exhibited long-lasting protection against immune-related disorders. System biology analysis has predicted an inverse correlation between Δpep27 immunization and gastroenteritis. Recently, we established that Δpep27-elicited Tregs repressed Wnt5a expression and enhanced barrier integrity, suggesting the restoration of immunological tolerance. Therefore, we evaluated whether Δpep27 can alleviate IBD. Δpep27 dose-dependent response was analyzed in dextran sulfate sodium-induced mice using transcriptome analysis. Pro- and anti-inflammatory signatures were cross-correlated by quantitative PCR and western blot analyses. To address the hierarchy regulating the activity of caspase-14, an undefined marker in IBD, and regulatory T cells (Tregs), antibody-based neutralization studies were conducted. Fecal microbiome profiles were analyzed by 16S rRNA pyrosequencing. Δpep27 significantly attenuated dextran sulfate sodium-induced oxidative stress parameters, proinflammatory cytokines, caspase-14 expression level, and upregulated tight junction, anti-inflammatory genes IL-10 and TGF-β1 via upregulation of Tregs to restore healthy gut microbiota. Neutralization studies unveiled that ∆pep27 had a remedial effect via Treg upregulation. Caspase-14, being an important mediator in the pathogenesis of IBD, can be an alternate therapeutic target in IBD. ∆pep27-increased Tregs repressed caspase-14 expression and reversed gut microbial dysbiosis, aiding to re-establish immunological tolerance.

Diabetes Mellitus is affecting people of all age groups worldwide. Many synthetic medicines available for type 2 diabetes mellitus in the market. However, there is a strong requirement for the development of better anti-diabetes compounds sourced especially from natural sources like medicinal plants. The extracts from the leaves of neem (Azadirachta indica) is traditionally known to have anti-diabetes properties. Therefore, there is an increased interest to identify potential compounds identified from neem leaf extracts showing predicted binding property with the known diabetes mellitus type 2 protein enzyme target phosphoenol-pyruvate carboxykinase(PEPCK). The structure data for compounds found in the leaf extract of neem was screened against PEPCK using molecular docking simulation and screening techniques. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses best binding properties with PEPCK. This observation finds application for further consideration in in vitro and in vivo validation.

Opioids are considered the gold standard therapy for pain. However, TLR-dependent negative effects in analgesia have highlighted the complexities in the pharmacodynamics of opioids. While successive studies have reported that morphine and Morphine-3-glucuronide (M3G) activate the TLR4 pathway, the structural details of this mechanism are lacking. Here, we have utilized various computational tools to reveal the structural dynamics of the opioid-bound TLR4/MD2 complex, and have proposed a potential TLR4 activation mechanism. Our results support previous findings, and include the novel insight that the stable binding of morphine and naloxone, but not M3G, in the MD2 cavity, is TLR4 dependent. Morphine interacts with MD2 near its Phe126 loop to induce the active conformation (MD2C); however, this binding is likely reversible, and the complex gains stability upon interaction with TLR4. M3G also induces the MD2C state, with both the Phe126 loop and the H1 loop being involved in MD2-M3G complex stability. Remarkably, naloxone, which requires TLR4 interaction for complex stability, switches the conformation of the gating loop to the inactive state (MD2°). Cumulatively, our findings suggest that ligand binding and receptor clustering occur successively in opioid-induced TLR4 signaling, and that MD2 plasticity and pocket hydrophobicity are crucial for the recognition and accommodation of ligands.

The high-risk human papillomavirus E6 (hrHPV E6) protein has been widely studied due to its implication in cervical cancer. In response to viral threat, activated kinases phosphorylate the IRF3 autoinhibitory domain, inducing type1 interferon production. HPV circumvents the antiviral response through the possible E6 interaction with IRF3 and abrogates p53's apoptotic activity by recruiting E6-associated protein. However, the molecular mechanism of IRF3 inactivation by hrHPV E6 has not yet been delineated. Therefore, we explored this mechanism through in silico examination of protein-protein and protein-ligand docking, binding energy differences, and computational alanine mutagenesis. Our results suggested that the LxxLL motifs of IRF3 binds within the hydrophobic pocket of E6, precluding Ser-patch phosphorylation, necessary for IRF3 activation and interferon induction. This model was further supported by molecular dynamics simulation. Furthermore, protein-ligand docking and drug resistance modeling revealed that the polar patches in the pocket of E6, which are crucial for complex stability and ligand binding, are inconsistent among hrHPV species. Such variabilities pose a risk of treatment failure owing to point mutations that might render drugs ineffective, and allude to multi-drug therapy. Overall, this study reveals a novel perspective of innate immune suppression in HPV infections and suggests a plausible therapeutic intervention.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-β and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo.

Capsular polysaccharides (CPS), a major virulence factor in Streptococcus pneumoniae, become thicker during blood invasion while not during asymptomatic nasopharyngeal colonization. However, the underlying mechanism controlling this differential pneumococcal CPS regulation remain unclear. Here, we show how VncR, the response regulator of the vancomycin resistance locus (vncRS operon), regulates CPS expression in vncR mutants in three serotype (type 2, 3, and 6B) backgrounds upon exposure to serum lactoferrin (LF). Comparative analysis of CPS levels in the wild type (WT) of three strains and their isogenic vncR mutants after LF exposure revealed a strain-specific alteration in CPS production. Consistently, VncR-mediated strain-specific CPS production is correlated with pneumococcal virulence, in vivo. Electrophoretic mobility-shift assay and co-immunoprecipitation revealed an interaction between VncR and the cps promoter (cpsp) in the presence of serum. In addition, in silico analysis uncovered this protein-DNA interaction, suggesting that VncR binds with the cpsp, and recognizes the strain-specific significance of the tandem repeats in cpsp. Taken together, the interaction of VncR and cpsp after serum exposure plays an essential role in regulating differential strain-specific CPS production, which subsequently determines strain-specific systemic virulence. This study highlights how host protein LF contributes to pneumococcal VncR-mediated CPS production. As CPS plays a significant role in immune evasion, these findings suggest that drugs designed to interrupt the VncR-mediated CPS production could help to combat pneumococcal infections.

TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed.