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1.
BMC Immunol ; 20(1): 48, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842739

RESUMO

BACKGROUND: Yersinia pestis, the etiological pathogen of plague, is capable of repressing the immune response of white blood cells to evade phagocytosis. The V-antigen (LcrV) was found to be involved in this process by binding to human Toll-like Receptor 2 (TLR2). The detailed mechanism behind this LcrV and TLR2 mediated immune response repression, however, is yet to be fully elucidated due to the lack of structural information. RESULTS: In this work, with protein structure modelling, we were able to construct a structure model of the heterotetramer of Y. pestis LcrV and human TLR2. Molecular dynamics simulation suggests the stability of this structure in aquatic environment. The LcrV model has a dumbbell-like structure with two globule domains (G1 at N-terminus and G2 away from membrane) connected with a coiled-coil linker (CCL) domain. The two horseshoe-shape TLR2 subunits form a V-shape structure, are not in direct contact with each other, and are held together by the LcrV homodimer. In this structure model, both the G1 and CCL domains are involved in the formation of LcrV homodimer, while all three domains are involved in LcrV-TLR2 binding. A mechanistic model was proposed based on this heterotetrameric structure model: The LcrV homodimer separates the TLR2 subunits to inhibit the dimerization of TLR2 and subsequent signal transfer for immune response; while LcrV could also inhibit the formation of heterodimers of TLR2 with other TLRs, and leads to immune response repression. CONCLUSIONS: A heterotetrameric structure of Y. pestis LcrV and human TLR2 was modelled in this work. Analysis of this modelled structure showed its stability in aquatic environments and the role of LcrV domains and residues in protein-protein interaction. A mechanistic model for the role of LcrV in Y. pestis pathogenesis is raised based on this heterotetrameric structure model. This work provides a hypothesis of LcrV function, with which further experimental validation may elucidate the role of LcrV in human immune response repression.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Complexos Multiproteicos/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/metabolismo , Domínio Catalítico , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
2.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569697

RESUMO

Cancer vaccine is a promising immunotherapeutic approach to train the immune system with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in cancer vaccines to mimic an infection process and amplify immune responses. The Toll-like receptor 2 and 6 (TLR2/TLR6) agonist dipalmitoyl-S-glyceryl cysteine (Pam2Cys) was demonstrated as an ideal candidate for synthetic vaccine adjuvants. However, the synthesis of Pam2Cys requires expensive N-protected cysteine as a key reactant, which greatly limits its application as a synthetic vaccine adjuvant in large-scaled studies. Here, we report the development of N-acetylated Pam2Cys analogs as TLR2/TLR6 agonists. Instead of N-protected cysteine, the synthesis utilizes N-acetylcysteine to bring down the synthetic costs. The N-acetylated Pam2Cys analogs were demonstrated to activate TLR2/TLR6 in vitro. Moreover, molecular docking studies were performed to provide insights into the molecular mechanism of how N-acetylated Pam2Cys analogs bind to TLR2/TLR6. Together, these results suggest N-acetylated Pam2Cys analogs as inexpensive and promising synthetic vaccine adjuvants to accelerate the development of cancer vaccines in the future.


Assuntos
Lipopeptídeos/química , Lipopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/química , Receptor 6 Toll-Like/agonistas , Receptor 6 Toll-Like/química , Humanos , Lipopeptídeos/síntese química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular
3.
Int J Biol Macromol ; 135: 1020-1027, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31152832

RESUMO

The effect of the ultrasonic treatment on the physicochemical properties, oil-holding capacities, foaming capacities, and TLR2-affinity of the polysaccharides from Pholiota nameko (PNPS), was evaluated. Compared with the protein content of PNPS before ultrasonic treatment, the protein content of PNPS all presented a decrease under different ultrasonic conditions. The viscosity of PNPS showed a decrease when the ultrasonic intensity was strong enough, as well as the molecular weight. The oil-holding capacity and the foaming capacity of PNPS showed a continuous increasing trend with the increase of the ultrasonic treatment time under a set ultrasonic power of 400 W. Further, the ultrasonic operation could induce the decrease of the affinity binding between PNPS and the receptor proteins TLR2. These data confirmed that applying the ultrasonic treatment could obtain PNPS with high carbohydrate contents, low viscosity and low TLR2-affinity under proper ultrasonic condition.


Assuntos
Fenômenos Químicos , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/isolamento & purificação , Extração Líquido-Líquido , Pholiota/química , Receptor 2 Toll-Like/química , Ondas Ultrassônicas , Ligação Proteica , Reologia , Receptor 2 Toll-Like/metabolismo
4.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1152-1159, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30684639

RESUMO

The nitrone spin trap 5,5­dimethyl­1­pyrroline N­oxide (DMPO) dampens endotoxin-induced and TLR4-driven priming of macrophages, but the mechanism remains unknown. The available information suggests a direct binding of DMPO to the TIR domain, which is shared between TLRs. However, TLR2-TIR domain is the only TLR that have been crystallized. Our in silico data show that DMPO binds to four specific residues in the BB-loop within the TLR2-TIR domain. Our functional analysis using hTLR2.6-expressing HEKs cells showed that DMPO can block zymosan-triggered-TLR2-mediated NF-κB activation. However, DMPO did not affect the overall TLR2-MyD88 protein-protein interaction. DMPO binds to the BB-loop in the TIR-domain and dampens downstream signaling without affecting the overall TIR-MyD88 interaction. These data encourage the use of DMPO-derivatives as potential mechanism-based inhibitors of TLR-triggered inflammation.


Assuntos
Óxidos N-Cíclicos/metabolismo , Inflamação/metabolismo , Óxidos de Nitrogênio/metabolismo , Transdução de Sinais , Marcadores de Spin , Receptor 2 Toll-Like/metabolismo , Animais , Óxidos N-Cíclicos/química , Células HEK293 , Humanos , Inflamação/imunologia , Camundongos , Simulação de Dinâmica Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Óxidos de Nitrogênio/química , Ligação Proteica , Domínios Proteicos , Células RAW 264.7 , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/química
5.
Biochem Biophys Res Commun ; 508(1): 152-158, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30471865

RESUMO

Our understanding of the PE/PPE family of proteins in M. tuberculosis (Mtb) pathogenesis is still evolving and their critical roles in the host immunomodulation are still in the discovery process. Earlier studies from our group have shown that TLR2-LRR domain plays an important role in regulating cytokine signalling by PPE proteins. The importance of TLR2-LRR domain 16-20 in the regulation of PPE17-induced pro-inflammatory signalling has been established recently. However, it is yet to find whether other PPE protein also targets the TLR2-LRR 16-20 domain for induction of pro-inflammatory responses. In the current study, we have explored the structural parameters and possible role of PPE65 in generating pro-inflammatory signalling molecules mediated through IRAK3 downstream of TLR2-LRR domain 16-20. This study conceptualizes the functional characteristics of PPE65 in infection condition and might possibly provide valuable information in exploring this protein as an immunomodulator in Mtb infection.


Assuntos
Proteínas de Bactérias/metabolismo , Inflamação/metabolismo , Mycobacterium tuberculosis/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Proteínas de Bactérias/química , Clonagem Molecular , Citocinas/análise , Citocinas/metabolismo , Microscopia Confocal , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Receptor 2 Toll-Like/química
6.
EMBO Rep ; 19(12)2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30337494

RESUMO

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.


Assuntos
Quitina/química , Quitina/metabolismo , Fungos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Receptor 2 Toll-Like/metabolismo , Animais , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitinases/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fatores Imunológicos/farmacologia , Ligantes , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células THP-1 , Receptor 1 Toll-Like/agonistas , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/química , Zimosan/metabolismo
7.
Nutrients ; 10(7)2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976865

RESUMO

Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam3CSK4. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam3CSK4-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam3CSK4, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam3CSK4-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam2CSK4-induced activation. Western blot results showed that phloretin significantly suppressed Pam3CSK4-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2⁻TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Floretina/farmacologia , Receptor 1 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Inflamação/metabolismo , Interleucina-8/metabolismo , Lipopeptídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Floretina/química , Floretina/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Receptor 1 Toll-Like/química , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Viral Immunol ; 31(4): 338-343, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29489437

RESUMO

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.


Assuntos
Anticorpos Neutralizantes/sangue , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C/imunologia , Receptor 2 Toll-Like/química , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/química , Adjuvantes Imunológicos/administração & dosagem , Animais , Linhagem Celular , Modelos Animais de Doenças , Hepatite C/sangue , Anticorpos Anti-Hepatite C/classificação , Humanos , Esquemas de Imunização , Lipopeptídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Organismos Livres de Patógenos Específicos , Receptor 2 Toll-Like/agonistas , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia
9.
Proteins ; 86(4): 475-490, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29383743

RESUMO

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.


Assuntos
Mutação Puntual , Multimerização Proteica , Receptores Toll-Like/química , Receptores Toll-Like/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Estabilidade Proteica , Receptor 1 Toll-Like/química , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
10.
Proteins ; 86(5): 524-535, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29383749

RESUMO

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.


Assuntos
Simulação de Dinâmica Molecular , Receptor 2 Toll-Like/química , Receptor 3 Toll-Like/química , Sítios de Ligação , Humanos , Fosforilação , Mutação Puntual , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Relação Estrutura-Atividade
11.
Int J Biol Macromol ; 109: 698-703, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29292152

RESUMO

Toll-like receptors (TLRs) encoded by the TLR multigene family play an important role in initial pathogen recognition in vertebrates. Among the TLRs, TLR2 and TLR4 may be of particular importance to reptiles. In order to study the evolutionary patterns and structural characteristics of TLRs, we explored the available genomes of several representative members of reptiles. 25 TLR2 genes and 19 TLR4 genes from reptiles were obtained in this study. Phylogenetic results showed that the TLR2 gene duplication occurred in several species. Evolutionary analysis by at least two methods identified 30 and 13 common positively selected codons in TLR2 and TLR4, respectively. Most positively selected sites of TLR2 and TLR4 were located in the Leucine-rich repeat (LRRs). Branch model analysis showed that TLR2 genes were under different evolutionary forces in reptiles, while the TLR4 genes showed no significant selection pressure. The different evolutionary adaptation of TLR2 and TLR4 among the reptiles might be due to their different function in recognizing bacteria. Overall, we explored the structure and evolution of TLR2 and TLR4 genes in reptiles for the first time. Our study revealed valuable information regarding TLR2 and TLR4 in reptiles, and provided novel insights into the conservation concern of natural populations.


Assuntos
Evolução Molecular , Duplicação Gênica , Genômica , Répteis/genética , Seleção Genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Animais , Genômica/métodos , Filogenia , Répteis/imunologia , Análise de Sequência de DNA , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/química
12.
J Leukoc Biol ; 103(2): 311-319, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29345364

RESUMO

Radioprotective 105 kDa (RP105, CD180) is a member of the Toll-like receptor (TLR) family that interacts with TLR2 and facilitates recognition of mature lipoproteins expressed by Mycobacterium tuberculosis and Mycobacterium bovis BCG. In this study, we used synthetic lipopeptide analogs of the M. tuberculosis 19 kDa lipoprotein to define structural characteristics that promote RP105-mediated host cell responses. A tripalmitoylated lipopeptide composed of the first 16 N-terminal amino acids of the M. tuberculosis 19 kDa lipoprotein induced RP105-dependent TNF and IL-6 production by macrophages. Di- and tripalmitoylated variants of this lipopeptide elicited an equivalent RP105-dependent response, indicating that while the lipid moiety is required for macrophage activation, it is not a determinant of RP105 dependency. Instead, substitution of two polar threonine residues at positions 7 and 8 with nonpolar alanine residues resulted in reduced RP105 dependency. These results strongly suggest that the amino acid composition of the M. tuberculosis 19 kDa lipoprotein, and likely other mycobacterial lipoproteins, is a key determinant of RP105 agonism.


Assuntos
Antígenos CD/metabolismo , Proteínas de Bactérias/farmacologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Lipopeptídeos/farmacologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Alanina/genética , Animais , Antígenos CD/química , Antígenos CD/genética , Proteínas de Bactérias/síntese química , Lipopeptídeos/síntese química , Lipopeptídeos/química , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/genética , Cultura Primária de Células , Treonina/genética , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Tuberculose/microbiologia
13.
Fish Shellfish Immunol ; 72: 629-638, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29183810

RESUMO

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.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência/veterinária , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia
14.
Arch Physiol Biochem ; 124(4): 326-329, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29160122

RESUMO

BACKGROUND: Soluble forms of Toll-like receptors (sTLR) 2 and 4 exert negative regulatory control on membrane-bound receptor activation. The study estimates the sTLR2 and sTLR4's serum levels in type 2 diabetes (T2D) and evaluates their relationship with metabolic and inflammatory parameters. SUBJECTS AND METHODS: Sixty three patients with T2D and 25 controls were enrolled. sTLR were assayed through ELISA. Inflammatory markers included Interleukin 6 (IL-6), high sensitivity C-reactive protein (hs-CRP) and tumour necrosis factor α. RESULTS: Analysis demonstrated lower sTLR2 level in T2D than in control subjects (1.15 ± 0.65 versus 1.44 ± 0.60 ng/ml, p = .019) while sTLR4 level remained similar (0.09 ± 0.16 versus 0.07 ± 0.12 ng/ml, p > .05) despite higher IL-6 (2.65 ± 2.46 versus 1.44 ± 0.22 pg/ml, p = .005) and hs-CRP (2.79 ± 2.89 versus 0.70 ± 0.89 mg/l, p < .001) concentrations. Neither sTLR correlated with BMI, HbA1c, plasma glucose and analysed cytokines (p > .05). CONCLUSION: The sTLR2 serum level in T2D patients was reduced despite elevated inflammatory parameters.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Regulação para Baixo , Receptor 2 Toll-Like/sangue , Biomarcadores/sangue , Bulgária , Proteína C-Reativa/análise , Estudos Transversais , Diabetes Mellitus Tipo 2/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hemoglobina A Glicada/análise , Hospitais Universitários , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Ambulatório Hospitalar , Solubilidade , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/sangue , Receptor 4 Toll-Like/química , Fator de Necrose Tumoral alfa/sangue , Regulação para Cima
15.
Biotech Histochem ; 92(7): 487-497, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28910171

RESUMO

We used immunohistochemistry to quantify and compare the expression of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in gingival tissues of both healthy individuals and patients with chronic periodontitis. We also correlated the expression of TLR2 and CD14 with the histological grades of chronic periodontitis. We examined 30 gingival specimens from chronic periodontitis patients and 10 from healthy individuals. Tissues from both groups were immunostained with antibodies against TLR2 and CD14. TLR2 and CD14 were expressed by endothelial cells, fibroblasts, lymphocytes and plasma cells. The immunohistochemical expression of TLR2 and CD14 was significantly greater in inflammatory cells of the chronic periodontitis group than in healthy individuals. Expression of these molecules was greater in the inflammatory cells of connective tissue adjacent to pocket epithelium in both groups. The expression of TLR2 and CD14 was greatest in the periodontitis group that was classified as severe grade, followed by moderate and mild grades, which suggests a role of TLR2 and CD14 in the pathogenesis of chronic periodontitis. The positive correlation of TLR2 and CD14 expression levels with the severity grades of chronic periodontitis suggests that they are correlated also with disease severity; therefore, they may be useful for predicting disease progression. Our findings are consistent with the possibility that CD14 acts as a co-receptor for TLR2.


Assuntos
Periodontite Crônica , Gengiva/química , Receptores de Lipopolissacarídeos/química , Receptor 2 Toll-Like/química , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Receptor 2 Toll-Like/metabolismo
16.
Sci Rep ; 7(1): 8363, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827637

RESUMO

Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that ß1ß2, loop-α3-loop, and α4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32ΔNß1ß2 significantly decreased the affinity to TLR2 while LipL32ΔCα4 slightly reduced it. Interestingly, LipL32ΔCenα3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cenα3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through Nß1ß2 and Cα4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Leptospira/imunologia , Lipoproteínas/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Lipoproteínas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Deleção de Sequência , Receptor 2 Toll-Like/química
17.
Biochim Biophys Acta Gen Subj ; 1861(11 Pt A): 2680-2689, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28734965

RESUMO

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.


Assuntos
Adjuvantes Imunológicos/química , Vacinas Anticâncer/química , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Receptor 2 Toll-Like/agonistas , Adjuvantes Imunológicos/isolamento & purificação , Adjuvantes Imunológicos/uso terapêutico , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Citocinas/biossíntese , Células Dendríticas/imunologia , Células HEK293 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Neoplasias/genética , Neoplasias/patologia , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/uso terapêutico , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Interface Usuário-Computador
18.
PLoS Pathog ; 13(4): e1006315, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28410407

RESUMO

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnß expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.


Assuntos
Amiloide/imunologia , Autoimunidade , Proteínas de Bactérias/imunologia , DNA Bacteriano/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Receptor 2 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Amiloide/química , Amiloide/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/genética , Receptor Toll-Like 9/química , Receptor Toll-Like 9/genética
19.
Anal Chem ; 89(9): 4882-4888, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28374588

RESUMO

Electrochemical detection of Pam3CSK4, a synthetic triacylated lipopeptide that mimics the structural moieties of its natural Gram negative bacterial pathogen-associated molecular pattern (PAMP) counterpart, has been achieved using hybridized toll-like receptors (TLR) combining TLR1 and TLR2 onto a single sensor surface. These sensors represent the first hybridized TLR sensors. The limit of detection for Pam3CSK4 attained was 7.5 µg/mL, which is within the same order of magnitude for that of the more labor-intensive and time-consuming cell-assay technique, 2.0 µg/mL. The results gathered in these electrochemical experiments show that sensors fabricated by immobilizing a mixture of cooperative TLR1 and -2 generate higher responses when exposed to the analyte in comparison to the control sensors fabricated using pure TLR1 or -2 standalone. A PAMP selectivity test was carried out in line with our inspiration from the mammalian innate immune response. TLRs1-5 as standalone biorecognition elements and the hybridized "TLR1 and 2" sensor surface were investigated, understanding the known TLR-PAMP interactions, through the exploitation of this electrochemical sensor fabrication technique. The experimental result is consistent with observations from previously published in vivo and in vitro studies, and it is the first demonstration of the simultaneous evaluation of electrochemical responses from multiple, unique fabricated TLR sensor surfaces against the same analyte.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Lipopeptídeos/análise , Animais , Limite de Detecção , Lipopeptídeos/química , Camundongos , Receptor 1 Toll-Like/química , Receptor 2 Toll-Like/química
20.
J Biol Chem ; 292(2): 652-660, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27909057

RESUMO

Ligand binding to Toll-like receptors (TLRs) results in dimerization of their cytosolic Toll/interleukin-1 receptor (TIR) domains and recruitment of post-receptor signal transducers into a complex signalosome. TLR activation leads to the production of transcription factors and pro-inflammatory molecules and the activation of phosphoinositide 3-kinases (PI3K) in a process that requires the multimodular B-cell adaptor for phosphoinositide 3-kinase (BCAP). BCAP has a sequence previously proposed as a "cryptic" TIR domain. Here, we present the structure of the N-terminal region of human BCAP and show that it possesses a canonical TIR fold. Dimeric BCAP associates with the TIR domains of TLR2/4 and MAL/TIRAP, suggesting that it is recruited to the TLR signalosome by multitypic TIR-TIR interactions. BCAP also interacts with the p85 subunit of PI3K and phospholipase Cγ, enzymes that deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), and these interactions provide a molecular explanation for BCAP-mediated down-regulation of inflammatory signaling.


Assuntos
Proteínas de Transporte/química , Multimerização Proteica , Transdução de Sinais , Receptor 2 Toll-Like/química , Receptor 4 Toll-Like/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/química , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fosfatidilinositol 3-Quinases , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Domínios Proteicos , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
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