ABSTRACT
Toll-like receptor 3 (TLR3) is an endosomal receptor involved in initiating immune responses upon viral infection by directly recognizing double-stranded RNA (dsRNA). As one of the most heavily glycosylated TLR family members, the role of glycan at N413 of TLR3 in ligand recognition has been in debate for decades. Herein, to investigate the role of glycans in TLR3, specifically at amino acid residue N413, molecular dynamic simulations were performed. The loop region of LRR12 (residues 323-355), which protrudes from the dsRNA binding TLR3 lateral surface was found to be vital for interacting with dsRNA via the formation of hydrogen bonds. The glycan at N413 not only prevented dsRNA from being exposed to the bulk water during the binding process but further stabilized dsRNA in the TLR3 binding site. When N413 was in the glycosylated form, the binding free energy of TLR3 interacting with dsRNA was significantly lower than that of TLR3 in the N413 unglycosylated form. Additionally, as the glycan at N413 functioned to alter the dynamics of the dsRNA binding process, its flexibility was meanwhile influenced by dsRNA. In all, these results demonstrate that the size, length, and branch of glycan at N413 affect the thermodynamics and dynamics of TLR3 recognition with dsRNA. This study further extends our understanding of the biological role of glycans in the innate immune recognition of dsRNA by TLR3 and provides a new perspective for modulating TLR3 function.
Subject(s)
Molecular Dynamics Simulation , Toll-Like Receptor 3 , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , RNA, Double-Stranded , Binding Sites , PolysaccharidesABSTRACT
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
Subject(s)
Famotidine/pharmacology , Histamine Antagonists/pharmacology , SARS-CoV-2/drug effects , Toll-Like Receptor 3/metabolism , A549 Cells , Binding Sites , Caco-2 Cells , Chemokine CCL2/metabolism , Coronavirus 3C Proteases/metabolism , HeLa Cells , Humans , Interferon Regulatory Factor-3/metabolism , Interleukin-6/metabolism , Molecular Docking Simulation , NF-kappa B/metabolism , Protein Binding , SARS-CoV-2/physiology , Signal Transduction , Toll-Like Receptor 3/chemistry , Virus ReplicationABSTRACT
Nucleic acid-sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B1) is indispensable for the trafficking of TLRs from the endoplasmic reticulum to the endosome, mechanisms of UNC93B1-mediated TLR regulation remain largely unknown. Here, we report two cryo-EM structures of human and mouse TLR3-UNC93B1 complexes and a human TLR7-UNC93B1 complex. UNC93B1 exhibits structural similarity to the major facilitator superfamily transporters. Both TLRs interact with the UNC93B1 amino-terminal six-helix bundle through their transmembrane and luminal juxtamembrane regions, but the complexes of TLR3 and TLR7 with UNC93B1 differ in their oligomerization state. The structural information provided here should aid in designing compounds to combat autoimmune diseases.
Subject(s)
Membrane Transport Proteins , Toll-Like Receptor 3 , Toll-Like Receptor 7 , Animals , Humans , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/ultrastructure , Mice , Protein Binding , Protein Multimerization , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/ultrastructure , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/ultrastructureABSTRACT
A new Cu(II) coordination polymer (CP) of [Cu5(µ3-OH)2(bcpt)4(bib)2] (1, bib = 1,4-bis(1-imidazoly)benzene and H2bcpt = 3,5-bis(3'-carboxyphenyl)-1,2,4-triazole) was synthesized by reaction of Cu(NO3)2·3H2O reacting with 3,5-bis(3'-carboxyphenyl)-1,2,4-triazole in the existence of 1,4-bis(1-imidazoly)benzene as the second ligand. The treatment activity of the compound on influenza A virus induced chronic obstructive pulmonary disease (COPD) was evaluated. First, the biological function of the lung was assessed by measuring the partial pressure for the carbon dioxide (PaCO2) and oxygen (PaO2) via the analysis of blood gas. Next, the inflammatory cytokines released by alveolar epithelial cells were determined via the ELISA test kit. In addition to this, the real-time RT-PCR was carried out to determine the inflammatory response relative expression in the alveolar epithelial cells. Finally, the relative expression of the TLR3 on the alveolar epithelial cells was revealed by western blot. Possible binding patterns were acquired from the post scoring software and molecular docking, which exhibited two possible functional side chain binding sites of TLR3 to compounds binding, possibly offering distinct regulatory mechanisms.
Subject(s)
Alveolar Epithelial Cells/chemistry , Influenza A virus , Pulmonary Disease, Chronic Obstructive , Epithelial Cells , Gene Expression , Humans , Ligands , Molecular Docking Simulation , Polymers/chemistry , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolismABSTRACT
Toll-like receptor 3 (TLR3) induces host innate immune response on recognition of viral double-stranded RNA (dsRNA). Although several studies of avian TLR3 have been reported, none of these studies used a gene knockout (KO) model to directly assess its role in inducing the immune response and effect on other dsRNA receptors. In this study, we determined the coding sequence of quail TLR3, identified isoforms, and generated TLR3 KO quail fibroblast (QT-35) cells using a CRISPR/Cas9 system optimized for avian species. The TLR3-mediated immune response was studied by stimulating the wild-type (WT) and KO QT-35 cells with synthetic dsRNA or polyinosinic:polycytidylic acid [poly(I:C)] or infecting the cells with different RNA viruses such as influenza A virus, avian reovirus, and vesicular stomatitis virus. The direct poly(I:C) treatment significantly increased IFN-ß and IL-8 gene expression along with the cytoplasmic dsRNA receptor, melanoma differentiation-associated gene 5 (MDA5), in WT cells, whereas no changes in all corresponding genes were observed in KO cells. We further confirmed the antiviral effects of poly(I:C)-induced TLR3-mediated immunity by demonstrating significant reduction of virus titer in poly(I:C)-treated WT cells, but not in TLR3 KO cells. On virus infection, varying levels of IFN-ß, IL-8, TLR3, and MDA5 gene upregulation were observed depending on the viruses. No major differences in gene expression level were observed between WT and TLR3 KO cells, which suggests a relatively minor role of TLR3 in sensing and exerting immune response against the viruses tested in vitro. Our data show that quail TLR3 is an important endosomal dsRNA receptor responsible for regulation of type I interferon and proinflammatory cytokine, and affect the expression of MDA5, another dsRNA receptor, most likely through cytokine-mediated communication.
Subject(s)
Birds , Immunity , Protein Isoforms , Toll-Like Receptor 3 , Animals , Birds/immunology , Cells, Cultured , Fibroblasts/immunology , Immunity/immunology , Poly I-C/pharmacology , Protein Isoforms/immunology , Quail/immunology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/immunologyABSTRACT
BACKGROUND: Coronavirus disease 2019 (COVID-19) linked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause severe illness and life-threatening pneumonia in humans. The current COVID-19 pandemic demands an effective vaccine to acquire protection against the infection. Therefore, the present study was aimed to design a multiepitope-based subunit vaccine (MESV) against COVID-19. METHODS: Structural proteins (Surface glycoprotein, Envelope protein, and Membrane glycoprotein) of SARS-CoV-2 are responsible for its prime functions. Sequences of proteins were downloaded from GenBank and several immunoinformatics coupled with computational approaches were employed to forecast B- and T- cell epitopes from the SARS-CoV-2 highly antigenic structural proteins to design an effective MESV. RESULTS: Predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (HLA) binding alleles, and collective global population coverage of 88.40%. Taken together, 276 amino acids long MESV was designed by connecting 3 cytotoxic T lymphocytes (CTL), 6 helper T lymphocyte (HTL) and 4 B-cell epitopes with suitable adjuvant and linkers. The MESV construct was non-allergenic, stable, and highly antigenic. Molecular docking showed a stable and high binding affinity of MESV with human pathogenic toll-like receptors-3 (TLR3). Furthermore, in silico immune simulation revealed significant immunogenic response of MESV. Finally, MEV codons were optimized for its in silico cloning into the Escherichia coli K-12 system, to ensure its increased expression. CONCLUSION: The MESV developed in this study is capable of generating immune response against COVID-19. Therefore, if designed MESV further investigated experimentally, it would be an effective vaccine candidate against SARS-CoV-2 to control and prevent COVID-19.
Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Humans , Immunogenicity, Vaccine/immunology , Molecular Docking Simulation , Pneumonia, Viral/immunology , SARS-CoV-2 , Sequence Analysis, Protein , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/immunology , Vaccines, Subunit/chemistry , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccinology/methods , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Vaccines/chemistry , Viral Vaccines/geneticsABSTRACT
The COVID-19 pandemic highlighted healthcare disparities in multiple countries. As such morbidity and mortality vary significantly around the globe between populations and ethnic groups. Underlying medical conditions and environmental factors contribute higher incidence in some populations and a genetic predisposition may play a role for severe cases with respiratory failure. Here we investigated whether genetic variation in the key genes for viral entry to host cells-ACE2 and TMPRSS2-and sensing of viral genomic RNAs (i.e., TLR3/7/8) could explain the variation in incidence across diverse ethnic groups. Overall, these genes are under strong selection pressure and have very few nonsynonymous variants in all populations. Genetic determinant for the binding affinity between SARS-CoV-2 and ACE2 does not show significant difference between populations. Non-genetic factors are likely to contribute differential population characteristics affected by COVID-19. Nonetheless, a systematic mutagenesis study on the receptor binding domain of ACE2 is required to understand the difference in host-viral interaction across populations.
Subject(s)
Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Toll-Like Receptors/genetics , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , Humans , Mutagenesis, Site-Directed , Protein Binding , Protein Domains , Selection, Genetic , Serine Endopeptidases/metabolism , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 8/genetics , Toll-Like Receptor 8/metabolism , Toll-Like Receptors/chemistry , Toll-Like Receptors/metabolism , Virus InternalizationABSTRACT
The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus 3C Proteases/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Amino Acid Sequence , Binding Sites , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , Cloning, Molecular/methods , Computational Biology/methods , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Genetic Vectors/chemistry , Genetic Vectors/immunology , HLA Antigens/chemistry , HLA Antigens/genetics , HLA Antigens/immunology , Humans , Immunity, Innate/drug effects , Immunogenicity, Vaccine , Interferon-gamma/genetics , Interferon-gamma/immunology , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , T-Lymphocytes, Cytotoxic/chemistry , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology , T-Lymphocytes, Helper-Inducer/chemistry , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/virology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , User-Computer Interface , Vaccines, SubunitABSTRACT
Toll-like receptor 3 (TLR3) provides the host with antiviral defense by initiating an immune signaling cascade for the production of type I interferons. The X-ray structures of isolated TLR3 ectodomain (ECD) and transmembrane (TM) domains have been reported; however, the structure of a membrane-solvated, full-length receptor remains elusive. We investigated an all-residue TLR3 model embedded inside a phospholipid bilayer using molecular dynamics simulations. The TLR3-ECD exhibited a ~30°-35° tilt on the membrane due to the electrostatic interaction between the N-terminal subdomain and phospholipid headgroups. Although the movement of dsRNA did not affect the dimer integrity of TLR3, its sugar-phosphate backbone was slightly distorted with the orientation of the ECD. TM helices exhibited a noticeable tilt and curvature but maintained a consistent crossing angle, avoiding the hydrophobic mismatch with the bilayer. Residues from the αD helix and the CD and DE loops of the Toll/interleukin-1 receptor (TIR) domains were partially absorbed into the lower leaflet of the bilayer. We found that the previously unknown TLR3-TIR dimerization interface could be stabilized by the reciprocal contact between αC and αD helices of one subunit and the αC helix and the BB loop of the other. Overall, the present study can be helpful to understand the signaling-competent form of TLR3 in physiological environments.
Subject(s)
Lipid Bilayers/chemistry , Phospholipids/chemistry , Toll-Like Receptor 3/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Phospholipids/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , Structure-Activity Relationship , Toll-Like Receptor 3/metabolismABSTRACT
Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-ß, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-ß by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer's disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.
Subject(s)
Antiviral Agents/metabolism , Astrocytes/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Phosphoproteins/metabolism , Syk Kinase/metabolism , Toll-Like Receptor 3/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Enzyme Activation/drug effects , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-7/metabolism , Ligands , Mice , NF-kappa B/metabolism , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Poly I-C/pharmacology , Protein Binding/drug effects , Signal Transduction/drug effects , Toll-Like Receptor 3/chemistry , Up-Regulation/drug effectsABSTRACT
Toll-like receptor 3 (TLR3), an endosomal receptor crucial for immune responses upon viral invasion. The TLR3 ectodomain (ECD) is responsible for double-stranded RNA (dsRNA) recognition and mutational analysis suggested that TLR3 ECD C-terminal dimerization is essential for dsRNA binding. Moreover, the L412F polymorphism of TLR3 is associated with human diseases. Although the mouse structure of the TLR3-dsRNA complex provides valuable insights, the structural dynamic behavior of the TLR3-dsRNA complex in humans is not completely understood. Hence, in this study, we performed molecular dynamic simulations of human wild-type and mutant TLR3 complexes. Our results suggested that apoTLR3 ECD dimers are unlikely to be stable due to the distance between the monomers are largely varied during simulations. The observed interaction energies and hydrogen bonds in dsRNA-bound TLR3 wild-type and mutant complexes indicate the presence of a weak dimer interface at the TLR3 ECD C-terminal site, which is required for effective dsRNA binding. The L412F mutant exhibited similar dominant motion compared to wild-type. Additionally, we identified the distribution of crucial residues for signal propagation in TLR3-dsRNA complex through the evaluation of residue betweenness centrality (CB). The results of this study extend our understanding of TLR3-dsRNA complex, which may assist in TLR3 therapeutics.
Subject(s)
Mutation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/metabolism , Binding Sites , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Models, Molecular , Molecular Dynamics Simulation , Principal Component Analysis , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Toll-Like Receptor 3/geneticsABSTRACT
Toll-like receptor 3 (TLR3), a pathogen recognition receptor of the innate immune response, recognizes and is activated by double-stranded RNA (dsRNA), which is indicative of viral exposure. A sensor design exercise was conducted, using surface plasmon resonance detection, through the examination of several immobilization approaches for TLR3 as a biorecognition element (BRE) onto a modified gold surface. To examine the TLR3-dsRNA interaction a synthetic analogue mimic, poly (I:C), was used. The interaction binding characteristics were determined and compared to literature data to establish the optimal immobilization method for the TLR3 BRE. A preliminary evaluation of the efficacy of the selected TLR3 surface as a broad-spectrum viral biosensor was also performed. Amine-coupling was found to be the most reliable method for manufacturing repeatable and consistent TLR3 BRE sensor surfaces, although this immobilization schema is not tailored to place the receptor in a spatially-specific orientation. The equilibrium dissociation constant (KD) measured for this immobilized TLR3-poly (I:C) interaction was 117⯱â¯3.30 pM. This evaluation included a cross-reactivity study using a selection of purified E. coli and synthetic double- and single-stranded nucleic acids. The results of this design exercise and ligand binding study will inform future work towards the development of a broad-spectrum viral sensor device.
Subject(s)
Biosensing Techniques/methods , Poly I-C/chemistry , Toll-Like Receptor 3/chemistry , Nucleic Acids , Protein Binding , Surface Plasmon ResonanceABSTRACT
Human T-lymphotropic virus (HTLV), the first human retrovirus has been discovered which is known to cause the age-old assassinating disease HTLV-1 associated myelopathy. Cancer caused by this virus is adult T cell leukemia/lymphoma which targets 10-20 million throughout the world. The effect of this virus extends to the fact that it causes chronic disease to the spinal cord resulting in loss of sensation and further causes blood cancer. So, to overcome the complications, we designed a subunit vaccine by the assimilation of B-cell, cytotoxic T-lymphocyte , and helper T-lymphocyte epitopes. The epitopes were joined together along with adjuvant and linkers and a vaccine was fabricated which was further subjected to 3D modeling. The physiochemical properties, allergenicity, and antigenicity were evaluated. Molecular docking and dynamics were performed with the obtained 3D model against toll like receptor (TLR-3) immune receptor. Lastly, in silico cloning was performed to ensure the expression of the designed vaccine in pET28a(+) expression vector. The future prospects of the study entailed the in vitro and in vivo experimental analysis for evaluating the immune response of the designed vaccine construct.
Subject(s)
Algorithms , Computational Biology/methods , Computer-Aided Design , Drug Design , Epitopes , HTLV-I Infections/prevention & control , Human T-lymphotropic virus 1/immunology , Viral Proteins/immunology , Viral Vaccines/pharmacology , HTLV-I Infections/immunology , HTLV-I Infections/virology , Human T-lymphotropic virus 1/genetics , Humans , Immunogenicity, Vaccine , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Structure, Tertiary , Structure-Activity Relationship , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/chemistry , Vaccines, Subunit/immunology , Vaccines, Subunit/pharmacology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Vaccines/chemistry , Viral Vaccines/genetics , Viral Vaccines/immunologyABSTRACT
Necroptosis, or caspase-independent programmed cell death, is known to be involved in various pathological conditions, such as ischemia/reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. Although several inhibitors of necroptosis have been identified, none of them are currently in clinical use. In the present study, we identified a new compound, 4-({[5-(4-aminophenyl)-4-ethyl-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-N-(1,3-thiazol-2-yl) benzamide (NTB451), with significant inhibitory activity on the necroptosis induced by various triggers, such as tumor necrosis factor-α (TNF-α) and toll-like receptor (TLR) agonists. Mechanistic studies revealed that NTB451 inhibited phosphorylation and oligomerization of mixed lineage kinase domain like (MLKL), and this activity was linked to its inhibitory effect on the formation of the receptor interacting serine/threonine-protein kinase 1 (RIPK1)-RIPK3 complex. Small interfering RNA (siRNA)-mediated RIPK1 knockdown, drug affinity responsive target stability assay, and molecular dynamics (MD) simulation study illustrated that RIPK1 is a specific target of NTB451. Moreover, MD simulation showed a direct interaction of NTB451 and RIPK1. Further experiments to ensure that the inhibitory effect of NTB451 was restricted to necroptosis and NTB451 had no effect on nuclear factor-κB (NF-κB) activation or apoptotic cell death upon triggering with TNF-α were also performed. Considering the data obtained, our study confirmed the potential of NTB451 as a new necroptosis inhibitor, suggesting its therapeutic implications for pathological conditions induced by necroptotic cell death.
Subject(s)
Apoptosis/drug effects , Imidazoles/pharmacology , Necrosis/metabolism , Dose-Response Relationship, Drug , Humans , Imidazoles/chemistry , Models, Molecular , Molecular Structure , NF-kappa B/metabolism , Necrosis/drug therapy , Phosphorylation/drug effects , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Structure-Activity Relationship , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The Toll-like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen-associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain-containing proteins. Mutations in the BB loop of the Toll-like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lpsd mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain-containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein-protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild-type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways.
Subject(s)
Point Mutation , Protein Multimerization , Toll-Like Receptors/chemistry , Toll-Like Receptors/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Maps , Protein Stability , Toll-Like Receptor 1/chemistry , Toll-Like Receptor 1/genetics , Toll-Like Receptor 1/metabolism , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/chemistry , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Toll-Like Receptors/metabolismABSTRACT
Extensive research performed on Toll-like receptor (TLR) signaling has identified residues in the Toll/interleukin-1 receptor (TIR) domains that are essential for its proper functioning. Among these residues, those in BB loop are particularly significant as single amino acid mutations in this region can cause drastic changes in downstream signaling. However, while the effect of these mutations on the function is well studied (like the P681H mutation in TLR2, the A795P mutation in TLR3, and the P714H mutation in TLR4), their influence on the dynamics and inter-residue networks is not well understood. The effects of local perturbations induced by these mutations could propagate throughout the TIR domain, influencing interactions with other TIR domain-containing proteins. The identification of these subtle changes in inter-residue interactions can provide new insights and structural rationale for how single-point mutations cause drastic changes in TIR-TIR interactions. We employed molecular dynamics simulations and protein structure network (PSN) analyses to investigate the structural transitions with special emphasis on TLR2 and TLR3. Our results reveal that phosphorylation of the Tyr 759 residue in the TIR domain of TLR3 introduces rigidity to its BB loop. Subtle differences in the intra BB loop hydrogen bonding network between TLR3 and TLR2 are also observed. The PSN analyses indicate that the TIR domain is highly connected and pinpoints key differences in the inter-residue interactions between the wild-type and mutant TIR domains, suggesting that TIR domain structure is prone to allosteric effects, consistent with the current view of the influence of allostery on TLR signaling.
Subject(s)
Molecular Dynamics Simulation , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 3/chemistry , Binding Sites , Humans , Phosphorylation , Point Mutation , Protein Binding , Protein Interaction Domains and Motifs , Signal Transduction , Structure-Activity RelationshipABSTRACT
Infectious Bursal Disease (IBD) is an acute, highly contagious and immunosuppressive disease of young chicken. The causative virus (IBDV) is a bi-segmented, double-stranded RNA virus. The virus encodes five major proteins, viral protein (VP) 1-5. VPs 1-3 have been characterized crystallographically. Albeit a rise in the number of studies reporting successful heterologous expression of VP5 in recent times, challenging the notion that rapid death of host cells overexpressing VP5 disallows obtaining sufficiently pure preparations of the protein for crystallographic studies, the structure of VP5 remains unknown and its function controversial. Our study describes the first 3D model of IBD VP5 obtained through an elaborate computational workflow. Based on the results of the study, IBD VP5 can be predicted to be a structural analog of the leucine-rich repeat (LRR) family of proteins. Functional implications arising from structural similarity of VP5 with host Toll-like receptor (Tlr) 3 also satisfy the previously reported opposing roles of the protein in first abolishing and later inducing host-cell apoptosis.
Subject(s)
Infectious bursal disease virus/chemistry , Toll-Like Receptor 3/chemistry , Viral Nonstructural Proteins/chemistry , Animals , Chickens , Gene Expression , Infectious bursal disease virus/genetics , Infectious bursal disease virus/isolation & purification , Infectious bursal disease virus/metabolism , Molecular Dynamics Simulation , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Toll-like receptors (TLRs) play important roles in fish innate immune and are involved in the defense process of bacteria invasion. In the present study, the full-length cDNA of TLR3 from the sea perch, Lateolabrax japonicus, was cloned and characterized. The full length of LjTLR3 cDNA was 3265 bp including an open reading frame of 2679 bp encoding a peptide of 922 amino acids. Tissues distribution analysis indicated that LjTLR3 showed a tissue-specific variation with high expression in spleen, head-kidney and liver. In order to investigate LjTLR3 functions against bacteria infection, the expression patterns of LjTLR3 after Vibrio harveyi and Streptococcus agalactiae challenge were detected by qRT-PCR, and the results showed that LjTLR3 was significant up-regulated after both bacteria stimulation in head-kidney, spleen and liver in a time-depended manner. Furthermore, the results by in situ hybridization experiments showed that positive signals of LjTLR3 mRNA in infected spleen and head-kidney were more numerous than that in the control group. In addition, intracellular localization revealed that LjTLR3 is distributed in the cytoplasm. In summary, these findings suggest that LjTLR3 was involved in the immune process under bacteria infection. This study would benefit to further clarify the roles of fish TLRs in the immune process and contribute to further study on enhancing disease resistance of L. japonicus.
Subject(s)
Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Perciformes/genetics , Perciformes/immunology , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/immunology , Amino Acid Sequence , Animals , Base Sequence , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling/veterinary , Phylogeny , Sequence Alignment/veterinary , Streptococcal Infections/immunology , Streptococcus agalactiae/physiology , Toll-Like Receptor 3/chemistry , Vibrio/physiology , Vibrio Infections/immunologyABSTRACT
Toll-like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV-2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response.
Subject(s)
Fish Diseases/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Regulation/immunology , Goldfish/genetics , Goldfish/immunology , Immunity, Innate/genetics , Amino Acid Sequence , Animals , Fish Proteins/chemistry , Gene Expression Profiling , Phylogeny , Sequence Alignment/veterinary , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/immunologyABSTRACT
Toll signaling is essential for expression of immune genes which are important for defense against bacterial, fungal and viral infections in invertebrates. Although several toll genes have been identified in the crustaceans, none of them has been investigated in freshwater crab Sinopotamon henanense. Moreover, the effect of cadmium (Cd) on toll gene expression has never been examined on the freshwater crabs which live in the sediments and are prone to heavy metal bioaccumulation. Our transcriptomic analysis of hepatopancreas tissue reveals that toll3 gene expression has been decreased when treated with Cd. In this study, we cloned one toll gene (hereby designated Shtoll3) from the crab. The full-length cDNA of Shtoll3 was 4488 bp, with an ORF of 3693 bp encoding a putative protein of 1230 amino acids, a 5'-untranslated region of 414 bp and a 3'-untranslated region of 781 bp. Phylogenetic analysis showed that ShToll3 was clustered into the group of DmToll8. The tissue distribution results showed that Shtoll3 was expressed widely in different tissues, with the highest in gills, and the lowest in hemocytes. Shtoll3 expression was down-regulated only in midguts after Aeromonas hydrophila infection. With Cd presence, Shtoll3 expression in response to A. hydrophila were up-regulated in midguts and gills, which was further confirmed by western blotting analysis. Moreover, the mRNA level of two antimicrobial peptides (AMPs) crustin and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll3, were analysised. Thus, we conclude that Cd changes the susceptibility of Shtoll3 to A. hydrophila infection in gills and midguts. This suggest that Shtoll3 may contribute to the innate immune defense of S. henanense to A. hydrophila and Cd can modify the immune function in epithelium.