RESUMEN
TLR5, which is activated by flagellin, plays an important role in initiating immune response to a broad spectrum of motile bacterial pathogens. TLRs induce intracellular signaling via dimerization of their TIR domains followed by adapter recruitment through multiple interactions of receptor and adapter TIRs. Here, a library of cell-permeable decoy peptides derived from the TLR5 TIR was screened for TLR5 signaling inhibition in the HEK-Blue-mTLR5 reporter cell line. The peptide demonstrating the strongest inhibition, 5R667, corresponded to the second helix of the region between the third and fourth ß-strands (helix Câ³). In addition to the TLR5-induced cytokine expression, 5R667 inhibited cytokine expression elicited by TLR4, TLR2, and TLR9. 5R667 also suppressed the systemic cytokine induction elicited by LPS administration in mice. 5R667 binding specificity was studied by time-resolved fluorescence spectroscopy in a cell-based assay. 5R667 demonstrated a multispecific binding pattern with respect to TIR domains: It bound TIRs of TLR adapters of the MyD88-dependent pathway, Toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP) and MyD88, and also the TIR of TLR5. TR667, the peptide derived from the TIRAP region, which is structurally homologous to 5R667, demonstrated binding and inhibitory properties similar to that of 5R667. The surface-exposed residues within TIR regions represented by 5R667 and TR667 form motifs, which are nearly 90% conserved in vertebrate evolution and are distinctive of TLR5 and TIRAP TIR domains. Thus, we have identified an evolutionary conserved adapter recruitment motif within TLR5 TIR, the function of which can be inhibited by selective cell-permeable decoy peptides, which can serve as pan-specific TLR inhibitors.
Asunto(s)
Factor 88 de Diferenciación Mieloide , Receptor Toll-Like 5 , Animales , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Péptidos/metabolismo , Citocinas/metabolismo , Receptores de Interleucina-1/metabolismoRESUMEN
Interaction of TLR9 with ligands activates NF-κB, leading to proinflammatory cytokine production. Excessive TLR activation is a pathogenic factor for inflammatory diseases. This study has examined cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain. CPDP 9R34, which included AB loop, ß-strand B, and N-terminal BB loop residues, inhibited TLR9 signaling most potently. CPDPs derived from α-helices C, D, and E (i.e., 9R6, 9R9, and 9R11) also inhibited TLR9-induced cytokines but were less potent than 9R34. 9R34 did not inhibit TLR2/1, TLR4, or TLR7 signaling. The N-terminal deletion modification of 9R34, 9R34-ΔN, inhibited TLR9 as potently as the full length 9R34. Binding of 9R34-ΔN to TIR domains was studied using cell-based Förster resonance energy transfer/fluorescence lifetime imaging approach. Cy3-labeled 9R34-ΔN dose-dependently decreased fluorescence lifetime of TLR9 TIR-Cerulean (Cer) fusion protein. Cy3-9R34-ΔN also bound TIRAP TIR, albeit with a lesser affinity, but not MyD88 TIR, whereas CPDP from the opposite TIR surface, 9R11, bound both adapters and TLR9. i.p. administration of 9R34-ΔN suppressed oligonucleotide-induced systemic cytokines and lethality in mice. This study identifies a potent, TLR9-specific CPDP that targets both receptor dimerization and adapter recruitment. Location of TIR segments that represent inhibitory CPDPs suggests that TIR domains of TLRs and TLR adapters interact through structurally homologous surfaces within primary receptor complex, leading to formation of a double-stranded, filamentous structure. In the presence of TIRAP and MyD88, primary complex can elongate bidirectionally, from two opposite ends, whereas in TIRAP-deficient cells, elongation is unidirectional, only through the αE side.
Asunto(s)
Dominios Proteicos/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 9/metabolismo , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Péptidos/metabolismoRESUMEN
TLRs sense a broad range of microbial molecules and initiate antimicrobial immune response. The members of the TLR family use cytoplasmic Toll/interleukin-1R homology (TIR) domain to initiate intracellular signaling. The activated TLRs dimerize their TIRs and recruit adapter proteins to the dimer, through multiple interactions of receptor and adapter TIR domains. Although TLRs play an essential role in innate immunity, the aberrant TLR signaling may cause pathogenic inflammation. This study has screened a library of cell-permeable decoy peptides (CPDPs) derived from the TLR7 TIR for interference with TLR7 signaling and identified new CPDPs that target the TLR7 signalosome assembly. Peptides 7R1, 7R6, 7R9, and 7R11 inhibited the TLR7-induced signaling in murine and human macrophages. The most potent inhibitory peptide of the four, 7R11, significantly reduced the systemic cytokine levels elicited by administration of a TLR7 agonist to mice. TLR7 TIR surface regions that correspond to inhibitory peptides generally corresponded to four TIR sites that mediate signalosome assembly for other TLRs. The cell-based Förster resonance energy transfer/fluorescence lifetime imaging confirmed that 7R9 and 7R11 interact with adapter TIRs. These findings clarify the molecular mechanisms that trigger the adapter recruitment to activated TLR7 and suggest that 7R9 and 7R11 have a significant translational potential as candidate or lead therapeutics for treatment of TLR7-related inflammatory diseases.
Asunto(s)
Citocinas/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/agonistas , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 7/agonistas , Animales , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Péptidos/química , Transducción de Señal/inmunología , Receptor Toll-Like 7/inmunologíaAsunto(s)
Péptidos/metabolismo , Dominios Proteicos/genética , Receptores Toll-Like/metabolismo , Animales , Permeabilidad de la Membrana Celular , Descubrimiento de Drogas , Humanos , Inmunidad Innata , Inmunomodulación , Péptidos/genética , Unión Proteica , Transducción de Señal , Receptores Toll-Like/genéticaRESUMEN
AIM: ß-Glucan is one of the most abundant polymers in nature and has been established as an immunomodulator. This compound has notable physiological effects on mammalian immune systems, including anti-tumor and anti-infective activities and can activate the immune response. It is considered that the immune-stimulating activities of ß-glucan can depend on physicochemical parameters, such as molecular size. Saccharomyces cerevisiae, also known as baker's yeast, is a frequently used source of ß-glucan. The aim of the experiments was to investigate how different Saccharomyces cerevisiae ß-glucan preparations with different molecular size affect interferon-gamma (IFN-γ) production in BALB/c mice. MATERIALS AND METHODS: In vivo and in vitro BALB/c mouse models were used for the investigations. Different ß-glucan preparations were orally administrated in the in vivo experiments. IFN-γ production in BALB/c mice was analyzed by enzyme-linked immunosorbent assay and measuring interferon-γ RNA concentration. RESULTS: The results showed that orally-administered ß-glucan from S. cerevisiae enhanced IFN-γ production in BALB/c mice in the in vivo model, but not by mouse leukocytes in vitro. Moreover, water-soluble ß-glucan enhanced IFN-γ production more effectively than did particulate ß-glucan. CONCLUSION: IFN-γ plays an important role in immunity against viral and bacterial infections. Our experiments have shown that ß-glucan preparations enhance IFN-γ production in BALB/c mice and can be potentially used for immune system stimulation in mammals. Current results may be used to develop soluble ß-glucan nutritional supplements.
Asunto(s)
Polisacáridos Fúngicos/farmacología , Factores Inmunológicos/farmacología , Interferón gamma/biosíntesis , beta-Glucanos/farmacología , Animales , Evaluación Preclínica de Medicamentos , Femenino , Interferón gamma/sangre , Ratones Endogámicos BALB C , Saccharomyces cerevisiae/químicaRESUMEN
A ß-1,3-endoglucanase produced by Streptomyces rutgersensis was purified to a homogeneity by the fractional precipitation with ammonium sulfate, ion exchange chromatography on Q-Sepharose and hydrophobic chromatography on Butyl Sepharose. A typical procedure provided 11.74-fold purification with 12.53 % yield. SDSPAGE of the purified protein showed one protein band. The exact molecular mass of the enzyme obtained by mass spectrometry was 41.25 kDa; the isoelectric point was between pH 4.24.4. The optimal ß-glucanase catalytic activity was at pH 7 and 50 °C. An enzyme was only active toward glucose polymers containing ß-1,3 linkages and hydrolyzed Saccharomyces cerevisiae cell wall ß-glucan in an endo-like way: reaction products were different molecular size ß-glucans, which were larger than glucose.