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Sample records for 2-macroglobulin receptor recognition

  1. Binding of receptor-recognized forms of alpha2-macroglobulin to the alpha2-macroglobulin signaling receptor activates phosphatidylinositol 3-kinase.

    PubMed

    Misra, U K; Pizzo, S V

    1998-05-29

    Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation. PMID:9593670

  2. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  3. alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)- dependent internalization of the urokinase receptor

    PubMed Central

    1995-01-01

    The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR- associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after

  4. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor.

    PubMed

    Segarini, P R; Nesbitt, J E; Li, D; Hays, L G; Yates, J R; Carmichael, D F

    2001-11-01

    Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

  5. Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein.

    PubMed

    Rebeck, G W; Harr, S D; Strickland, D K; Hyman, B T

    1995-02-01

    Both apolipoprotein E and its receptor, the low-density-lipoprotein receptor-related protein (LRP), are associated with senile plaques in Alzheimer's disease. We examined the relationship of other LRP-related molecules to senile plaques. LRP is a multifunctional receptor that binds and rapidly internalizes at least seven ligands: apolipoprotein E, activated alpha 2-macroglobulin, tissue and urokinase-type plasminogen activators, plasminogen activator inhibitor-1, lipoprotein lipase, and lactoferrin. Using immunohistochemistry, we showed that all of these ligands, representing a diverse group of otherwise apparently unrelated proteins, accumulate on senile plaques. We also studied expression of the receptor-associated protein, a physiological inhibitor of LRP, in the hippocampal formation from normal subjects and Alzheimer's disease patients. Receptor-associated protein colocalizes with LRP on neuronal soma, but not on neuronal processes or reactive astrocytes. It is not present on senile plaques. These results suggest that senile plaque-associated LRP can bind its ligands, but clearance of these compounds may be impaired in the vicinity of senile plaques.

  6. Upregulation of capsule enables Streptococcus pyogenes to evade immune recognition by antigen-specific antibodies directed to the G-related alpha2-macroglobulin-binding protein GRAB located on the bacterial surface.

    PubMed

    Dinkla, Katrin; Sastalla, Inka; Godehardt, Antonia W; Janze, Nina; Chhatwal, Gursharan S; Rohde, Manfred; Medina, Eva

    2007-07-01

    One of the major problems associated with the development of a vaccine against Streptococcus pyogenes is the ability of this pathogen to escape recognition by antibodies directed against conserved surface-associated determinants and to establish infection in the setting of an acquired immune response. Identification of the mechanism(s) used by S. pyogenes to avoid recognition by antigen-specific antibodies and escape killing in blood was the focus of this study. We showed here that S. pyogenes was capable of surviving in human blood containing high levels of antibodies directed against the G-related alpha2-macroglobulin-binding protein GRAB, a highly conserved bacterial surface protein. S. pyogenes upregulated the hyaluronic acid capsule production during incubation with human blood, suggesting that the capsule may structurally minimize antibody access to protein GRAB. This hypothesis was confirmed by the ability of anti-GRAB antibodies to promote opsonophagocytosis of a capsule-deficient mutant of S. pyogenes but not of the encapsulated wild-type strain. Capsule upregulation and protection of S. pyogenes from opsonophagocytosis in the presence of anti-GRAB antibodies was also observed in a murine model of streptococcal infection. Thus, masking of surface immunogenic determinants by the hyaluronic acid capsule may constitute a novel mechanism of S. pyogenes for evasion of antigen-specific antibodies.

  7. Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.

    PubMed

    Wyatt, Amy R; Kumita, Janet R; Farrawell, Natalie E; Dobson, Christopher M; Wilson, Mark R

    2015-01-01

    Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein. PMID:26103636

  8. Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge.

    PubMed

    Luo, Sheng-Wei; Cai, Luo; Qi, Zeng-Hua; Wang, Cong; Liu, Yuan; Wang, Wei-Na

    2015-08-01

    The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge. PMID:25917969

  9. Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge.

    PubMed

    Luo, Sheng-Wei; Cai, Luo; Qi, Zeng-Hua; Wang, Cong; Liu, Yuan; Wang, Wei-Na

    2015-08-01

    The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge.

  10. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  11. Cultured human monocytes synthesize and secrete alpha2-macroglobulin

    PubMed Central

    1977-01-01

    alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a radioimmunoassay. Unfractionated mononuclear leukocytes produced greater amounts of the macroglobulin (4.0 vs. 0.8 ng/10(6) cells) than did subpopulations enriched in T or B+T lymphocytes, by passage through nylon wool or cotton wool columns, respectively. Still higher concentrations of alpha2-macroglobulin (40 ng/10(6) cells) were measured in the supernates of glass-adherent mononuclear leukocyte cultures. These results suggest that cells of monocyte-macrophage lineage are mainly, if not exclusively, responsible for the appearance of alpha2- macroglobulin in the supernate of human peripheral blood leukocyte cultures. The de novo synthesis and release of alpha2- macroglobulin by cultured monocytes was demonstrated by immunoprecipitation of radioactivity from supernates of 32S-methionine- labeled glass-adherent cells. Antiserum against purified alpha2- macroglobulin was used in both Ouchterlony double diffusion and double antibody precipitation tests. SDS-polyacrylamide gel electrophoresis of immunoprecipitates showed that most of the radioactivity comigrated with authentic alpha2-macroglobulin subunit at about 160,000 daltons. PMID:68095

  12. Characterization of alpha-2-macroglobulin from groupers.

    PubMed

    Chuang, Wen-Hsiao; Lee, Kuo-Kau; Liu, Ping-Chung

    2013-08-01

    Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma

  13. Inactivation of α2-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

    PubMed Central

    Croisier, J.-L.; Camus, G.; Brumioul, D.; Mathy-Hartert, M.; Sondag, D.; Deby, C.; Lamy, M.

    1994-01-01

    The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active α2- macroglobulin (80 μg/ml) totally inhibits the proteolytic activity of trypsin (14 μg/ml) by trapping this protease. But after a 20 min incubation of α2-macroglobulin at 37°C with 2 × 106 human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10−7 M) and cytochalasin B (10−8 M), 100% of trypsin activity was recovered, indicating a total inactivation of α2-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates α2-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10−7 M also inactivates α2-macroglobulin, which indicates that an important cause of α2-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase. PMID:18472929

  14. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-2-macroglobulin immunological test system. 866.5620 Section 866.5620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the alpha-2-macroglobulin (a serum protein) in plasma. Measurement of alpha-2-macroglobulin may aid...

  15. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5620 Alpha-2-macroglobulin immunological test system. (a) Identification. An alpha-2-macroglobulin... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-2-macroglobulin immunological test...

  16. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5620 Alpha-2-macroglobulin immunological test system. (a) Identification. An alpha-2-macroglobulin... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-2-macroglobulin immunological test...

  17. Synthesis of alpha 2-macroglobulin in rat hepatocytes and in a cell-free system.

    PubMed

    Andus, T; Gross, V; Tran-Thi, T A; Heinrich, P C

    1983-01-10

    The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000. PMID:6186524

  18. Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

    PubMed

    Andus, T; Ramadori, G; Heinrich, P C; Knittel, T; Meyer zum Büschenfelde, K H

    1987-11-01

    Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. PMID:2444437

  19. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-2-macroglobulin immunological test system. 866.5620 Section 866.5620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5620 Alpha-2-macroglobulin...

  20. Role of zinc and alpha2 macroglobulin on thymic endocrine activity and on peripheral immune efficiency (natural killer activity and interleukin 2) in cervical carcinoma.

    PubMed

    Mocchegiani, E; Ciavattini, A; Santarelli, L; Tibaldi, A; Muzzioli, M; Bonazzi, P; Giacconi, R; Fabris, N; Garzetti, G G

    1999-01-01

    Decreased natural killer (NK) activity as well as interleukin 2 (IL-2) are risk factors for the progression of cervical carcinoma. NK activity and IL-2 may be thymus controlled. Plasma levels of active thymulin, a zinc-dependent thymic hormone (ZnFTS), are reduced in cancer because of the low peripheral zinc bioavailability. Zinc and thymulin are relevant for normal immune functions. Alpha2-macroglobulin is an inhibitor of matrix metalloproteases (MMPs) against invasive tumour proliferation. Because alpha2-macroglobulin has a binding affinity (Kd) for zinc that is higher than does thymulin, it may play a key role in immune efficiency in cancer. Plasma samples of 22 patients (age range 35-60 years) with locally advanced squamous cervical carcinoma and with FIGO stage Ib2-IIb were examined. They showed reduced active thymulin, decreased NK activity and IL-2 production, increased soluble IL-2 receptor (sIL-2R) and augmented alpha2-macroglobulin in the circulation, whereas plasma zinc levels were within the normal range for age. Significant positive correlations were found between zinc or active thymulin and alpha2-macroglobulin (r = 0.75, P < 0.01, r = 0.78, P < 0.01, respectively) in cancer patients. In vitro zinc increases IL-2 production from peripheral blood mononuclear cells (PBMCs) of cancer patients. These data suggest that an increase in alpha2-macroglobulin, which competes with thymulin for zinc binding, may be involved in causing a thymulin deficit with a consequent decrease of IL-2 and NK cytotoxicity. Thus, physiological zinc treatment in cervical carcinoma maybe restores impaired central and peripheral immune efficiency.

  1. Role of zinc and α2macroglobulin on thymic endocrine activity and on peripheral immune efficiency (natural killer activity and interleukin 2) in cervical carcinoma

    PubMed Central

    Mocchegiani, E; Ciavattini, A; Santarelli, L; Tibaldi, A; Muzzioli, M; Bonazzi, P; Giacconi, R; Fabris, N; Garzetti, G G

    1999-01-01

    Decreased natural killer (NK) activity as well as interleukin 2 (IL-2) are risk factors for the progression of cervical carcinoma. NK activity and IL-2 may be thymus controlled. Plasma levels of active thymulin, a zinc-dependent thymic hormone (ZnFTS), are reduced in cancer because of the low peripheral zinc bioavailability. Zinc and thymulin are relevant for normal immune functions. α2-Macroglobulin is an inhibitor of matrix metalloproteases (MMPs) against invasive tumour proliferation. Because α2-macroglobulin has a binding affinity (Kd) for zinc that is higher than does thymulin, it may play a key role in immune efficiency in cancer. Plasma samples of 22 patients (age range 35–60 years) with locally advanced squamous cervical carcinoma and with FIGO stage Ib2–IIb were examined. They showed reduced active thymulin, decreased NK activity and IL-2 production, increased soluble IL-2 receptor (sIL-2R) and augmented α2-macroglobulin in the circulation, whereas plasma zinc levels were within the normal range for age. Significant positive correlations were found between zinc or active thymulin and α2-macroglobulin (r = 0.75, P< 0.01, r = 0.78, P< 0.01, respectively) in cancer patients. In vitro zinc increases IL-2 production from peripheral blood mononuclear cells (PBMCs) of cancer patients. These data suggest that an increase in α2-macroglobulin, which competes with thymulin for zinc binding, may be involved in causing a thymulin deficit with a consequent decrease of IL-2 and NK cytotoxicity. Thus, physiological zinc treatment in cervical carcinoma maybe restores impaired central and peripheral immune efficiency. © 1999 Cancer Research Campaign PMID:9888464

  2. Quantification of functional and inactivated alpha 2-macroglobulin in sepsis.

    PubMed

    Abbink, J J; Nuijens, J H; Eerenberg, A J; Huijbregts, C C; Strack van Schijndel, R J; Thijs, L G; Hack, C E

    1991-01-23

    Alpha 2-macroglobulin (alpha 2 M) in vitro inhibits numerous proteinases that are generated during inflammatory reactions and therefore, probably plays an important role in diseases such as sepsis. To monitor the state of alpha 2 M in sepsis, we developed novel assays for functional and inactive alpha 2M. Functional alpha 2M in plasma was measured by quantitating the binding of alpha 2M to solid-phase trypsin. Inactive alpha 2M (i alpha 2M) was assessed with a monoclonal antibody, mcAb M1, that specifically reacts with a neodeterminant exposed on i alpha 2M. This mcAb in combination with chromogenic substrates was used to detect alpha 2M-proteinase complexes. Functional alpha 2M was reduced in plasma from 48 patients with clinical sepsis compared to healthy controls (p less than 0.0001). Levels of functional alpha 2M on admission and the lowest levels encountered in 23 patients with shock were lower than in 25 normotensive patients (p = 0.023 and p = 0.009, respectively). Increased levels of i alpha 2M (greater than 30 nM) at least on one occasion were found in only 4 of the 48 patients, being not different in hypotensive compared with normotensive patients, and not in patients who died compared with those who survived. Levels of functional alpha 2M correlated significantly with levels of factor XII and prekallikrein suggesting that decreases in alpha 2M at least in part were due to contact activation. Indeed, in two patients with increased i alpha 2M, complexes between alpha 2M and kallikrein were demonstrated in addition to plasmin- and thrombin-alpha 2M complexes.

  3. Binding of transforming growth factor-beta (TGF-beta) to pregnancy zone protein (PZP). Comparison to the TGF-beta-alpha 2-macroglobulin interaction.

    PubMed

    Philip, A; Bostedt, L; Stigbrand, T; O'Connor-McCourt, M D

    1994-04-15

    Pregnancy zone protein (PZP) is quantitatively the most important pregnancy-associated plasma protein and it has strong similarity to alpha 2-macroglobulin. Since alpha 2-macroglobulin is a binding protein for transforming growth factors-beta (TGF-beta), it was of interest to test whether the related protein, PZP, also binds to these growth-regulatory proteins. Using affinity-labelling methods, we demonstrate that PZP binds both TGF-beta 1 and TGF-beta 2 and that the binding characteristics are similar to those of the TGF-beta-alpha 2-macroglobulin interaction. TGF-beta 2 and TGF-beta 1 bind to PZP in a predominantly noncovalent manner in vitro. TGF-beta 1 and TGF-beta 2 bind to both the dimeric and tetrameric forms of PZP. Our studies also indicate that PZP binds TGF-beta 2 with higher affinity than TGF-beta 1. Finally, we demonstrate that PZP inhibits the binding of TGF-beta 1 and TGF-beta 2 to their cell surface receptors. The increased level of PZP during pregnancy may affect the action of TGF-beta by regulating the distribution, clearance and/or general availability of TGF-beta. The preferential binding of TGF-beta 2 over TGF-beta 1 by PZP implies that PZP may differentially regulate the action of TGF-beta 1 and TGF-beta 2.

  4. BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells.

    PubMed

    Wakle-Prabagaran, Monali; Lorca, Ramón A; Ma, Xiaofeng; Stamnes, Susan J; Amazu, Chinwendu; Hsiao, Jordy J; Karch, Celeste M; Hyrc, Krzysztof L; Wright, Michael E; England, Sarah K

    2016-04-19

    The large-conductance, voltage-gated, calcium (Ca(2+))-activated potassium channel (BKCa) plays an important role in regulating Ca(2+)signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCain myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCaand both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCain an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca(2+)to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca(2+)-free medium and inhibitors of various Ca(2+)signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca(2+)through store-operated Ca(2+)channels. Finally, we found that the specific BKCablocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCachannel in human myometrium and that BKCaand its immunomodulatory interacting partners regulate Ca(2+)dynamics in hMSMCs during pregnancy. PMID:27044074

  5. Plant pattern-recognition receptors.

    PubMed

    Zipfel, Cyril

    2014-07-01

    Plants are constantly exposed to would-be pathogens in their immediate environment. Yet, despite relying on innate immunity only, plants are resistant to most microbes. They employ pattern-recognition receptors (PRRs) for sensitive and rapid detection of the potential danger caused by microbes and pests. Plant PRRs are either surface-localized receptor kinases (RKs) or receptor-like proteins (RLPs) containing various ligand-binding ectodomains that perceive pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). In this review, I summarize our current knowledge of plant PRRs and their ligands, illustrating the multiple molecular strategies employed by plant PRRs to activate innate immune signaling to survive.

  6. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

    PubMed

    Fyfe, Cameron D; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W; Cogdell, Richard J; Wall, Daniel M; Burchmore, Richard J S; Byron, Olwyn; Walker, Daniel

    2015-07-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  7. The elimination of alpha 2-macroglobulin complexes from the arthritic joint. An experimental study in dogs.

    PubMed

    Ekerot, L; Ohlsson, K

    1982-01-01

    The polyvalent protease inhibitor, alpha 2-macroglobulin, may counteract a protease-mediated rheumatoid joint destruction. The elimination of the complexed inhibitor from joints was analysed in inflamed and noninflamed conditions of the knee joints in dogs. The arthritis was immunologically induced. The fate of intra-articularly injected radioactive alpha 2-macroglobulin complexes was studied by external measurements, analyses of blood, lymph and urine, and by autoradiographic and immunohistologic methods. The results indicate that the elimination of complexes was accelerated by inflammation and joint movements with a half-life shorter than 2 hours in acute arthritis. In addition to absorption into the synovial membrane and degradation in macrophage-like cells, the process of clearance included elimination of complexes via the blood and the lymph capillaries of the joint and subsequent degradation in cells belonging to the reticuloendothelial system in the lymph nodes and the liver. The degradation products were excreted in increasing amounts in the urine. Referring to the earlier recognized high degree of saturation of synovial fluid alpha 2-macroglobulin in rheumatoid arthritis, the finding of a rapid articular clearance of alpha 2-macroglobulin complexes suggests a pronounced release of endoproteases under clinical conditions. PMID:6186018

  8. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-2-macroglobulin immunological test system. 866.5620 Section 866.5620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the diagnosis of blood-clotting or clot lysis disorders. (b) Classification. Class II...

  9. Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

    PubMed Central

    Doan, Ninh; Gettins, Peter G. W.

    2007-01-01

    Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein. PMID:17608619

  10. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  11. α-2-Macroglobulin in Saliva Is Associated with Glycemic Control in Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Aitken, Juan Pablo; Ortiz, Carolina; Morales-Bozo, Irene; Rojas-Alcayaga, Gonzalo; Baeza, Mauricio; Beltran, Caroll

    2015-01-01

    Background. Subjects with type 2 diabetes mellitus (DM2) require an adequate glycemic control to avoid diabetic complications. Currently, saliva biomarkers are used as a diagnostic tool and can be indicative of the degree of progression and control of various diseases. Several studies indicate that α-2-macroglobulin levels are elevated in diabetic patients. Methods. 120 subjects with DM2 were enrolled and classified into two groups according to their glycemic control (percentage of glycated hemoglobin-A1c (HbA1c), <7% adequate glycemic control group; >7% inadequate glycemic control group). The relationship between α-2-macroglobulin levels from saliva samples and HbA1c was subsequently evaluated. Results. We found a positive correlation between α-2-macroglobulin and HbA1c (r = 0.778 and P < 0.0001). Area under the receivers operating characteristic (ROC) curve of α-2-macroglobulin indicated a positive discrimination threshold of α-2-macroglobulin (AUC = 0.903, CI 95%: 0.847–0.959, P < 0.0001) to diagnose glycemic control. Conclusions. Our data strongly suggest that the level of saliva α-2-macroglobulin is an indicator for the degree of glycemic control in diabetic patients and represents a promising alternative method to evaluate this parameter. PMID:25821337

  12. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  13. [Progress of pattern recognition receptors of molluscs].

    PubMed

    Gao, Qian; Zhao, Qin-ping; Ma, Xiao-xue; Dong, Hui-fen

    2015-08-01

    Molluscs have established complete innate immunity to defense against pathogens. The pattern recognition receptors (PRRs) are the sensory receptors of molluscs to resist outside invaders, as the first reactor to initiate the innate immune response. Some PRRs have been identified in several molluscs, including Toll-like receptors (TLRs) , C-type lectins, galectins, lipopolysaccharide-β-1,3-glucan binding protein (LGBP), Clq domain-containing protein (ClqDC), and peptidoglycan recognition protein (PGRP). PRRs have various biological activities and play important roles in the defense system of molluscs. This paper reviews the research progress of PRRs in molluscs.

  14. Pattern recognition receptors in antifungal immunity.

    PubMed

    Plato, Anthony; Hardison, Sarah E; Brown, Gordon D

    2015-03-01

    Receptors of the innate immune system are the first line of defence against infection, being able to recognise and initiate an inflammatory response to invading microorganisms. The Toll-like (TLR), NOD-like (NLR), RIG-I-like (RLR) and C-type lectin-like receptors (CLR) are four receptor families that contribute to the recognition of a vast range of species, including fungi. Many of these pattern recognition receptors (PRRs) are able to initiate innate immunity and polarise adaptive responses upon the recognition of fungal cell wall components and other conserved molecular patterns, including fungal nucleic acids. These receptors induce effective mechanisms of fungal clearance in normal hosts, but medical interventions, immunosuppression or genetic predisposition can lead to susceptibility to fungal infections. In this review, we highlight the importance of PRRs in fungal infection, specifically CLRs, which are the major PRR involved. We will describe specific PRRs in detail, the importance of receptor collaboration in fungal recognition and clearance, and describe how genetic aberrations in PRRs can contribute to disease pathology.

  15. Evolution of alpha 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human alpha 2-macroglobulin.

    PubMed Central

    Starkey, P M; Barrett, A J

    1982-01-01

    Plasma or serum samples from a large number of vertebrate species were screened for the presence of a papain-binding protein resembling human alph a 2-macroglobulin (alpha 2M). The screening method depended on the unique property of alpha 2M of binding proteinases in such a way that the enzyme retains partial activity against low-molecular-weight substrates. A papain-binding protein was detected in serum from members of all the major vertebrate taxa. In mammals, birds, reptiles and amphibians the protein had an Mr similar to that of human alpha 2M (725 000), but in fish, including dipnoans, actinopterygians, elasmobranchs and cyclostomes, the papain-binding protein was of Mr about 360 000. Of the invertebrate species tested, all of which were arthropods, two were negative, but the horseshoe crab, an arachnid, did possess a papain-binding protein, although this was heterogeneous in electrophoresis and differed from alpha 2M in resisting inactivation by methylamine. From the results, and a detailed study of the properties of the fish papain-binding protein described in an accompanying paper [Starkey, Fletcher & Barrett (1982) Biochem. J. 205, 97-104], it seems that alpha 2M first appeared in an ancestor of all modern vertebrates as a protein of Mr 360 000 and that the larger macroglobulin (Mr 725 000) first appeared in an ancestor of the tetrapods. Images Fig. 1. PMID:6181778

  16. Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

    PubMed

    Borisova, Elena A; Gorbushin, Alexander M

    2014-08-01

    We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa. PMID:24830774

  17. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

    PubMed Central

    Carlsson, J; Herrmann, B F; Höfling, J F; Sundqvist, G K

    1984-01-01

    Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin. Images PMID:6198282

  18. Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition

    PubMed Central

    Garcia-Ferrer, Irene; Arêde, Pedro; Gómez-Blanco, Josué; Luque, Daniel; Duquerroy, Stephane; Castón, José R.; Goulas, Theodoros; Gomis-Rüth, F. Xavier

    2015-01-01

    The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric “snap trap.” PMID:26100869

  19. Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition.

    PubMed

    Garcia-Ferrer, Irene; Arêde, Pedro; Gómez-Blanco, Josué; Luque, Daniel; Duquerroy, Stephane; Castón, José R; Goulas, Theodoros; Gomis-Rüth, F Xavier

    2015-07-01

    The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼ 180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric "snap trap."

  20. Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra.

    PubMed

    Qian, Jing; Ren, Chunhua; Xia, Jianjun; Chen, Ting; Yu, Zonghe; Hu, Chaoqun

    2016-07-10

    The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra. PMID:27033585

  1. An alpha-2 macroglobulin in the pearl oyster Pinctada fucata: Characterization and function in hemocyte phagocytosis of Vibrio alginolyticus.

    PubMed

    Wang, Zhongliang; Wang, Bei; Chen, Gang; Lu, Yishan; Jian, Jichang; Wu, Zaohe

    2016-08-01

    Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond. PMID:27346151

  2. An alpha-2 macroglobulin in the pearl oyster Pinctada fucata: Characterization and function in hemocyte phagocytosis of Vibrio alginolyticus.

    PubMed

    Wang, Zhongliang; Wang, Bei; Chen, Gang; Lu, Yishan; Jian, Jichang; Wu, Zaohe

    2016-08-01

    Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond.

  3. Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra.

    PubMed

    Qian, Jing; Ren, Chunhua; Xia, Jianjun; Chen, Ting; Yu, Zonghe; Hu, Chaoqun

    2016-07-10

    The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.

  4. [Uniform method for determining the alpha 1-antitrypsin and alpha 2-macroglobulin activity in human blood serum (plasma)].

    PubMed

    Nartikova, V F; Paskhina, T S

    1979-01-01

    A modified spectrophotometric method is developed for simultaneous estimation of alpha 1-antitrypsin and alpha 2-macroglobulin in human blood serum (plasma); the method is based on dissimilar interaction of these inhibitors with trypsin in the systems with a low molecular substrate N-alpha-benzoyl-l-arginine ethyl ester. alpha 1-Antitrypsin was estimated by inhibition of the arginine esterase activity of trypsin in a mixture containing human blood serum diluted 50-fold. alpha 2-Macroglobulin was estimated by maintained arginine esterase activity of the trypsin-alpha 2-macroglobulin complex, formed after interaction of an excess of trypsin with blood serum, diluted 10-fold and after subsequent inactivation of free, unbound with alpha 2-macroglobulin, trypsin by treatment with the soy bean inhibitor of trypsin. alpha 1-Antitrypsin and alpha 2-macrog-obulin were estimated by means of the method described in blood serum of healthy persons and in patients with burns or with carcinoma of pancreas. The method enables to estimate two main inhibitors of blood plasma proteinases in a small volume of blood serum (0.1 ml) very rapidly and specifically using commercially available substrate; the method might be recommended for routine clinical analysis.

  5. Pattern-recognition receptors in pulp defense.

    PubMed

    Staquet, M-J; Carrouel, F; Keller, J-F; Baudouin, C; Msika, P; Bleicher, F; Kufer, T A; Farges, J-C

    2011-07-01

    Initial sensing of infection is mediated by germline-encoded pattern-recognition receptors (PRRs), the activation of which leads to the expression of inflammatory mediators responsible for the elimination of pathogens and infected cells. PRRs act as immune sensors that provide immediate cell responses to pathogen invasion or tissue injury. Here, we review the expression of PRRs in human dental pulp cells, namely, receptors from the Toll-like (TLR) and Nod-like NLR families, by which cells recognize bacteria. Particular attention is given to odontoblasts, which are the first cells encountered by pathogens and represent, in the tooth, the first line of defense for the host. Understanding cellular and molecular mechanisms associated with the recognition of bacterial pathogens by odontoblasts is critical for the development of therapeutic strategies that aim at preventing excessive pulp inflammation and related deleterious effects.

  6. Pattern-recognition receptors in human eosinophils

    PubMed Central

    Kvarnhammar, Anne Månsson; Cardell, Lars Olaf

    2012-01-01

    The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1–5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1β, interleukin-6, tumour necrosis factor-α and granulocyte–macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively. PMID:22242941

  7. Pattern-recognition receptors in human eosinophils.

    PubMed

    Kvarnhammar, Anne Månsson; Cardell, Lars Olaf

    2012-05-01

    The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1β, interleukin-6, tumour necrosis factor-α and granulocyte-macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.

  8. Pattern recognition receptors in microbial keratitis.

    PubMed

    Taube, M-A; del Mar Cendra, M; Elsahn, A; Christodoulides, M; Hossain, P

    2015-11-01

    Microbial keratitis is a significant cause of global visual impairment and blindness. Corneal infection can be caused by a wide variety of pathogens, each of which exhibits a range of mechanisms by which the immune system is activated. The complexity of the immune response to corneal infection is only now beginning to be elucidated. Crucial to the cornea's defences are the pattern-recognition receptors: Toll-like and Nod-like receptors and the subsequent activation of inflammatory pathways. These inflammatory pathways include the inflammasome and can lead to significant tissue destruction and corneal damage, with the potential for resultant blindness. Understanding the immune mechanisms behind this tissue destruction may enable improved identification of therapeutic targets to aid development of more specific therapies for reducing corneal damage in infectious keratitis. This review summarises current knowledge of pattern-recognition receptors and their downstream pathways in response to the major keratitis-causing organisms and alludes to potential therapeutic approaches that could alleviate corneal blindness.

  9. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    PubMed

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

  10. Differential transcription of multiple forms of alpha-2-macroglobulin in carp (Cyprinus carpio) infected with parasites.

    PubMed

    Onara, Dalia F; Forlenza, Maria; Gonzalez, Santiago F; Rakus, Krzysztof Ł; Pilarczyk, Andrzej; Irnazarow, Ilgiz; Wiegertjes, Geert F

    2008-01-01

    Alpha-2-macroglobulin (a2M) is a non-specific protease inhibitor involved in host defense mechanisms, inhibiting both endogenous and exogenous proteases. It is unique among the plasma anti-proteases with respect to the diversity of proteases that it can inactivate. Carp a2M consists of an alpha and beta chain of which the first includes the bioactive regions. Previously, three a2M alpha chain sequences were reported for East-Asian common carp. We studied a2M alpha chain variability in European common carp and report the cloning of a fourth a2M alpha chain with distinct sequence diversity in the bait region. The role of a2M in the immune response to parasites was studied in the liver of carp infected with Trypanoplasma borreli or with Ichthyophthirius multifiliis. Quantitative gene transcription analysis showed a differential regulation of the four isoforms, most clearly seen in infections with I. multifiliis. A2M3 was the only a2M isoform with a highly upregulated transcription during infection, suggesting that this particular isoform is of foremost biological importance.

  11. Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells.

    PubMed Central

    Willingham, M C; Yamada, S S; Pastan, I

    1978-01-01

    We have been developing a procedure for localizing intracellular antigens in cultured cells, by using peroxidase-labeled antibodies, that allows good morphologic preservation. Although useful, our previous technique did not preserve the morphology of membranes, and the location of the peroxidase reaction product was difficult to establish. In this paper, we report major improvements on the basic technique that markedly enhance the quality of localization and of morphology. Saponin is used to permeabilize membranes without destroying their morphology. The amount of reaction product is enhanced with a peroxidase-antiperoxidase label. The clarity of morphologic detail and contrast of reaction product density are increased by using postsectioning staining with the osmium/thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures. We have applied this technique to the ultrastructural localization of alpha2-macroglobulin and demonstrated that it is localized in membrane-limited vesicles. We have also used this method to improve the preservation of structures for localization by fluorescence microscopy. Images PMID:81488

  12. Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response.

    PubMed

    Federici Canova, Donata; Pavlov, Anton M; Norling, Lucy V; Gobbetti, Thomas; Brunelleschi, Sandra; Le Fauder, Pauline; Cenac, Nicolas; Sukhorukov, Gleb B; Perretti, Mauro

    2015-11-10

    Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. PMID:26385167

  13. Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study.

    PubMed

    Rehman, Ahmed Abdur; Ahsan, Haseeb; Khan, Fahim Halim

    2016-02-01

    A α2M homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α2M was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep α2M was found to be made up of two fragments of 102 and 31 kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79Ǻ, which makes it much more compact than its human homologue. It entraps only 1 mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures--enthalpy change (ΔH), entropy change (ΔS) and Gibb's free energy change (ΔG), along with number of binding sites (N) and affinity constant (K) are explored for α2M-trypsin binding for the first time for any known α2M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin-α2M interaction is enthalpy driven event.

  14. Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response

    PubMed Central

    Canova, Donata Federici; Pavlov, Anton M.; Norling, Lucy V.; Gobbetti, Thomas; Brunelleschi, Sandra; Le Fauder, Pauline; Cenac, Nicolas; Sukhorukov, Gleb B.; Perretti, Mauro

    2015-01-01

    Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. PMID:26385167

  15. Copper is taken up efficiently from albumin and alpha2-macroglobulin by cultured human cells by more than one mechanism.

    PubMed

    Moriya, Mizue; Ho, Yi-Hsuan; Grana, Anne; Nguyen, Linh; Alvarez, Arrissa; Jamil, Rita; Ackland, M Leigh; Michalczyk, Agnes; Hamer, Pia; Ramos, Danny; Kim, Stephen; Mercer, Julian F B; Linder, Maria C

    2008-09-01

    Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and alpha(2)-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3-6 muM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from alpha(2)-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65-80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100-500 microM) inhibited copper uptake from albumin by 20-30% in both cell types and that from alpha(2)-macroglobulin by 0-30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. alpha(2)-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified.

  16. Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

    PubMed Central

    Huang, Michael Li-Hsuan; Austin, Christopher J. D.; Sari, Marie-Agnès; Suryo Rahmanto, Yohan; Ponka, Prem; Vyoral, Daniel; Richardson, Des R.

    2013-01-01

    Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225–6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1−/− and Lrp1+/+ cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1−/− and Lrp1+/+ cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down

  17. Pattern-recognition receptors and gastric cancer.

    PubMed

    Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O; Mitchell, Hazel M

    2014-01-01

    Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy.

  18. Pattern-Recognition Receptors and Gastric Cancer

    PubMed Central

    Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O.; Mitchell, Hazel M.

    2014-01-01

    Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy

  19. Serum alpha 2-macroglobulin in haemodialysis patients: baseline and kinetic studies.

    PubMed

    Argilés, A; Kerr, P G; Mourad, G; Mion, C M; Atkins, R C

    1993-01-01

    The pathogenesis of dialysis related amyloidosis remains unresolved despite the identification of beta 2-microglobulin (beta 2M) as the major protein constituent, as well as other proteins being present in the deposits. Among the latter we have assessed the serum concentrations of alpha 2-macroglobulin (alpha 2M) both in the baseline stage and during the haemodialysis (HD) procedure. We have also assessed the influence of the membrane on alpha 2M kinetics. Fifteen HD patients with histologically proven dialysis-related amyloidosis (DRA group) and 15 HD patients clinically and radiologically considered dialysis-related amyloidosis free (control group) were included in the baseline study. Blood was sampled the day before the second dialysis of the week and alpha 2M, beta 2M and alpha 1 antitrypsin were determined along with the routine biological analysis of these patients. Serum alpha 2M was greater in dialysis-related amyloidosis than in control patients (t = 2.35; P < 0.026). Serum beta 2M was similar in both groups. The serum alpha 2M and beta 2M correlated in patients with dialysis-related amyloidosis (r = 0.64; P < 0.01), while no correlation was found in controls (r = 0.17; NS). Stepwise analysis taking the presence of dialysis-related amyloidosis as the dependent variable retained the serum alpha 2M concentration as the first variable in the model (F = 4.4; partial r = 0.38; P < 0.046).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7505905

  20. Activated α2-Macroglobulin Binding to Human Prostate Cancer Cells Triggers Insulin-like Responses

    PubMed Central

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2015-01-01

    Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2–3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2–3-fold increase in lipogenesis as determined by 6-[14C]glucose or 1-[14C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [14CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy. PMID:25720493

  1. Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies

    PubMed Central

    2014-01-01

    Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general. PMID:25428871

  2. Predominant role of neutrophils in the inactivation of alpha 2-macroglobulin in arthritic joints.

    PubMed

    Abbink, J J; Kamp, A M; Nieuwenhuys, E J; Nuijens, J H; Swaak, A J; Hack, C E

    1991-09-01

    We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in

  3. Expression of pattern recognition receptors in cholesteatoma.

    PubMed

    Lee, Ho Yun; Park, Moon Suh; Byun, Jae Yong; Kim, Young Il; Yeo, Seung Geun

    2014-02-01

    Although many immunologic mechanisms have been investigated in studies of the pathogenesis of cholesteatoma, the role of pattern recognition receptors (PRRs) has not been fully determined. Therefore, we assessed innate immune responses in patients with cholesteatoma. We prospectively evaluated 21 patients with acquired cholesteatoma between August 2010 and July 2012. Cholesteatoma specimens were obtained during surgery, and skin from the external meatus of each patient was used as a control. RNA was extracted from these tissue samples, followed by real-time PCR to quantitatively assess the relative expression of toll-like receptors (TLRs), NOD-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I, NO synthase (NOS) and cytokines. The levels of TLR-2, -3, -4, -6, -7, and -10, NOD-2, and IL-1 and -8 mRNAs were significantly higher in the cholesteatoma than in the skin specimens (p < .05). The expression levels of TLR-2 and -3, RIG-I, IL-6, and TNF-α mRNAs were significantly higher in cholesteatomas from women than from men. The levels of TLR-8, NOD-2, IL-12, and TNF-α mRNAs were significantly higher in recurrent than in initial cholesteatoma specimens (p < .05). Hearing level did not correlate with the levels of expression of mRNAs encoding TLRs, NLRs, NOS, RIG-I and related cytokines (p > .05). In conclusion, alterations in innate immunity triggered by PRRs are important in the pathophysiology of cholesteatoma. Gender differences and frequency of surgery may affect the expression of PRRs in cholesteatomas.

  4. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

    PubMed Central

    Barrett, A J; Brown, M A; Sayers, C A

    1979-01-01

    alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not

  5. Pattern recognition receptors in innate immunity, host defense, and immunopathology.

    PubMed

    Suresh, Rahul; Mosser, David M

    2013-12-01

    Infection by pathogenic microbes initiates a set of complex interactions between the pathogen and the host mediated by pattern recognition receptors. Innate immune responses play direct roles in host defense during the early stages of infection, and they also exert a profound influence on the generation of the adaptive immune responses that ensue. An improved understanding of the pattern recognition receptors that mediate innate responses and their downstream effects after receptor ligation has the potential to lead to new ways to improve vaccines and prevent autoimmunity. This review focuses on the control of innate immune activation and the role that innate immune receptors play in helping to maintain tissue homeostasis.

  6. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  7. Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks.

    PubMed

    Sieckmann, Donna G; Jaffe, Howard; Golech, Susanne; Cai, DeCheng; Hallenbeck, John M; McCarron, Richard M

    2014-09-15

    Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed (3)H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation.

  8. Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif.

    PubMed

    Jensen, Gitte A; Andersen, Olav M; Bonvin, Alexandre M J J; Bjerrum-Bohr, Ida; Etzerodt, Michael; Thøgersen, Hans C; O'Shea, Charlotte; Poulsen, Flemming M; Kragelund, Birthe B

    2006-09-29

    The low-density lipoprotein receptor-related protein (LRP) interacts with more than 30 ligands of different sizes and structures that can all be replaced by the receptor-associated protein (RAP). The double module of complement type repeats, CR56, of LRP binds many ligands including all three domains of RAP and alpha2-macroglobulin, which promotes the catabolism of the Abeta-peptide implicated in Alzheimer's disease. To understand the receptor-ligand cross-talk, the NMR structure of CR56 has been solved and ligand binding experiments with RAP domain 1 (RAPd1) have been performed. From chemical shift perturbations of both binding partners upon complex formation, a HADDOCK model of the complex between CR56 and RAPd1 has been obtained. The binding residues are similar to a common binding motif suggested from alpha2-macroglobulin binding studies and provide evidence for an understanding of their mutual cross-competition pattern. The present structural results convey a simultaneous description of both binding partners of an LRP-ligand complex and open a route to a broader understanding of the binding specificity of the LRP receptor, which may involve a general four-residue receptor-ligand recognition motif common to all LRP ligands. The present result may be beneficial in the design of antagonists of ligand binding to the LDL receptor family, and especially of drugs for treatment of Alzheimer's disease.

  9. α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes.

    PubMed

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J; Bandoh, Betty; Barfod, Lea; Cavanagh, David R; Andersen, Gregers R; Hviid, Lars

    2015-07-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M--(and IgM-) binding domains of PfEMP1

  10. The structural basis for receptor recognition of human interleukin-18

    SciTech Connect

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.

  11. The structural basis for receptor recognition of human interleukin-18

    DOE PAGESBeta

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; et al

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  12. The structural basis for receptor recognition of human interleukin-18

    PubMed Central

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-01-01

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity. PMID:25500532

  13. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization.

    PubMed

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam

    2014-02-01

    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  14. The role of pattern recognition receptors in the innate recognition of Candida albicans.

    PubMed

    Zheng, Nan-Xin; Wang, Yan; Hu, Dan-Dan; Yan, Lan; Jiang, Yuan-Ying

    2015-01-01

    Candida albicans is both a commensal microorganism in healthy individuals and a major fungal pathogen causing high mortality in immunocompromised patients. Yeast-hypha morphological transition is a well known virulence trait of C. albicans. Host innate immunity to C. albicans critically requires pattern recognition receptors (PRRs). In this review, we summarize the PRRs involved in the recognition of C. albicans in epithelial cells, endothelial cells, and phagocytic cells separately. We figure out the differential recognition of yeasts and hyphae, the findings on PRR-deficient mice, and the discoveries on human PRR-related single nucleotide polymorphisms (SNPs).

  15. Perirhinal Cortex Muscarinic Receptor Blockade Impairs Taste Recognition Memory Formation

    ERIC Educational Resources Information Center

    Gutierrez, Ranier; De la Cruz, Vanesa; Rodriguez-Ortiz, Carlos J.; Bermudez-Rattoni, Federico

    2004-01-01

    The relevance of perirhinal cortical cholinergic and glutamatergic neurotransmission for taste recognition memory and learned taste aversion was assessed by microinfusions of muscarinic (scopolamine), NMDA (AP-5), and AMPA (NBQX) receptor antagonists. Infusions of scopolamine, but not AP5 or NBQX, prevented the consolidation of taste recognition…

  16. Pattern Recognition Receptors in Innate Immunity, Host Defense, and Immunopathology

    ERIC Educational Resources Information Center

    Suresh, Rahul; Mosser, David M.

    2013-01-01

    Infection by pathogenic microbes initiates a set of complex interactions between the pathogen and the host mediated by pattern recognition receptors. Innate immune responses play direct roles in host defense during the early stages of infection, and they also exert a profound influence on the generation of the adaptive immune responses that ensue.…

  17. Emerging principles governing signal transduction by pattern-recognition receptors.

    PubMed

    Kagan, Jonathan C; Barton, Gregory M

    2014-11-13

    The problem of recognizing and disposing of non-self-organisms, whether for nutrients or defense, predates the evolution of multicellularity. Accordingly, the function of the innate immune system is often intimately associated with fundamental aspects of cell biology. Here, we review our current understanding of the links between cell biology and pattern-recognition receptors of the innate immune system. We highlight the importance of receptor localization for the detection of microbes and for the initiation of antimicrobial signaling pathways. We discuss examples that illustrate how pattern-recognition receptors influence, and are influenced by, the general membrane trafficking machinery of mammalian cells. In the future, cell biological analysis likely will rival pure genetic analysis as a tool to uncover fundamental principles that govern host-microbe interactions.

  18. Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

    PubMed

    de Boer, J P; Creasey, A A; Chang, A; Abbink, J J; Roem, D; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-12-01

    Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis.

  19. Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human alpha2-macroglobulin and rabbit immunoglobulin.

    PubMed

    Souza, C T; Moura, M B; Magalhaes, A; Heneine, L G; Olortegui, C C; Diniz, C R; Sanchez, E F

    2001-09-01

    Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees. PMID:11544086

  20. Alpha2-macroglobulin from an Atlantic shrimp: biochemical characterization, sub-cellular localization and gene expression upon fungal challenge.

    PubMed

    Perazzolo, Luciane Maria; Bachère, Evelyne; Rosa, Rafael Diego; Goncalves, Priscila; Andreatta, Edemar Roberto; Daffre, Sirlei; Barracco, Margherita Anna

    2011-12-01

    In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70-90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection. PMID:21888978

  1. Alpha2-macroglobulin from an Atlantic shrimp: biochemical characterization, sub-cellular localization and gene expression upon fungal challenge.

    PubMed

    Perazzolo, Luciane Maria; Bachère, Evelyne; Rosa, Rafael Diego; Goncalves, Priscila; Andreatta, Edemar Roberto; Daffre, Sirlei; Barracco, Margherita Anna

    2011-12-01

    In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70-90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection.

  2. Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

    PubMed Central

    de Boer, J P; Creasey, A A; Chang, A; Abbink, J J; Roem, D; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-01-01

    Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis. PMID:7693593

  3. The diversity of receptor recognition in cholesterol-dependent cytolysins.

    PubMed

    Tabata, Atsushi; Ohkura, Kazuto; Ohkubo, Yukimasa; Tomoyasu, Toshifumi; Ohkuni, Hisashi; Whiley, Robert A; Nagamune, Hideaki

    2014-03-01

    Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59.

  4. Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma

    PubMed Central

    Thieme, René; Kurz, Susanne; Kolb, Marlen; Debebe, Tewodros; Holtze, Susanne; Morhart, Michaela; Huse, Klaus; Szafranski, Karol; Platzer, Matthias; Hildebrandt, Thomas B.; Birkenmeier, Gerd

    2015-01-01

    Background The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. Conclusion We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti

  5. Pyrrolic tripodal receptors for the molecular recognition of carbohydrates: ditopic receptors for dimannosides.

    PubMed

    Francesconi, Oscar; Nativi, Cristina; Gabrielli, Gabriele; Gentili, Matteo; Palchetti, Marco; Bonora, Beatrice; Roelens, Stefano

    2013-08-26

    Synthetic ditopic receptors, designed for the molecular recognition of dimannosides, have been prepared by bridging two monotopic units effectively recognizing mannosides with linkers of the appropriate size and flexibility, endowed with hydrogen-bonding groups. Affinities toward the α and β glycosides of the biologically relevant Manα(1-2)Man disaccharide were measured by NMR spectroscopy and isothermal titration calorimetry (ITC) in polar organic media (30-40 % DMF in chloroform). Significant selectivities and affinities in the micromolar range were observed in most cases, with two newly designed receptors being the most effective receptors of the set, together with a distinct preference of the dimannosides for the (S) enantiomer of the receptor in all cases. A 3D view of the recognition mode was elucidated by a combined NMR spectroscopic/molecular modeling approach, showing the dimannoside included in the cleft of the receptor. Compared to the monotopic precursors, the ditopic receptors showed markedly improved recognition properties, proving the efficacy of the modular receptor design for the recognition of disaccharides.

  6. Pattern recognition receptors and central nervous system repair.

    PubMed

    Kigerl, Kristina A; de Rivero Vaccari, Juan Pablo; Dietrich, W Dalton; Popovich, Phillip G; Keane, Robert W

    2014-08-01

    Pattern recognition receptors (PRRs) are part of the innate immune response and were originally discovered for their role in recognizing pathogens by ligating specific pathogen associated molecular patterns (PAMPs) expressed by microbes. Now the role of PRRs in sterile inflammation is also appreciated, responding to endogenous stimuli referred to as "damage associated molecular patterns" (DAMPs) instead of PAMPs. The main families of PRRs include Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs), AIM2-like receptors (ALRs), and C-type lectin receptors. Broad expression of these PRRs in the CNS and the release of DAMPs in and around sites of injury suggest an important role for these receptor families in mediating post-injury inflammation. Considerable data now show that PRRs are among the first responders to CNS injury and activation of these receptors on microglia, neurons, and astrocytes triggers an innate immune response in the brain and spinal cord. Here we discuss how the various PRR families are activated and can influence injury and repair processes following CNS injury.

  7. Host defence against Candida albicans and the role of pattern-recognition receptors.

    PubMed

    Gauglitz, Gerd G; Callenberg, Helene; Weindl, Günther; Korting, Hans C

    2012-05-01

    Recognition of Candida albicans is mediated by several classes of pattern-recognition receptors, including Toll-like receptors and C-type lectin receptors. Cell wall components of C. albicans, interact with the pattern-recognition receptors, which are expressed by different cells, primarily antigen-presenting cells. This review aims to discuss the different pattern-recognition receptors responsible for recognition of special structures of C. albicans, which are known to activate intracellular signals that finally lead to directed and efficient host defence.

  8. Molecular Recognition of Natural Products by Resorc[4]arene Receptors.

    PubMed

    D'Acquarica, Ilaria; Ghirga, Francesca; Quaglio, Deborah; Cerreto, Antonella; Ingallina, Cinzia; Tafi, Andrea; Botta, Bruno

    2016-01-01

    This review is aimed at providing an overview of the up-to-now published literature on resorc[4]arene macrocycles exploited as artificial receptors for the molecular recognition of some classes of natural products. A concise illustration of the main synthetic strategies developed to afford the resorc[4]arene scaffold is followed by a report on the principles of the gas-phase investigation of recognition phenomena by mass spectrometry (MS). Emphasis is placed on gas-phase studies of diastereoisomeric complexes generated inside a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer by resorc[4]arene receptors towards a series of natural products, namely amino acids, amphetamine, ethanolamine neurotransmitters, dipeptides, vinca alkaloids and nucleosides. The literature outcomes discussed here, taken largely from our own revisited work, have been completed by references to other studies, in order to draw a broader picture of this rapidly evolving field of research. PMID:26654589

  9. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    PubMed

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  10. Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition

    NASA Astrophysics Data System (ADS)

    Hakoshima, Toshio; Murase, Kohji; Hirano, Yoshinori; Sun, Tai-Ping

    Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

  11. New insights into TRP channels: Interaction with pattern recognition receptors.

    PubMed

    Han, Huirong; Yi, Fan

    2014-01-01

    An increasing number of studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms in response to pathogens or tissue injury, which is performed via germ-line encoded pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) or dangers-associated molecular patterns (DAMPs). Intracellular pathways linking immune and inflammatory response to ion channel expression and function have been recently identified. Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge about classifications, functions, and interactions of TRP channels and PRRs, which may provide new insights into their roles in the pathogenesis of inflammatory diseases.

  12. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  13. Pattern Recognition Receptors in Cancer Progression and Metastasis

    PubMed Central

    Pandey, Sanjay; Singh, Saurabh; Anang, Vandana; Bhatt, Anant N; Natarajan, K; Dwarakanath, Bilikere S

    2015-01-01

    The innate immune system is an integral component of the inflammatory response to pathophysiological stimuli. Toll-like receptors (TLRs) and inflammasomes are the major sensors and pattern recognition receptors (PRRs) of the innate immune system that activate stimulus (signal)-specific pro-inflammatory responses. Chronic activation of PRRs has been found to be associated with the aggressiveness of various cancers and poor prognosis. Involvement of PRRs was earlier considered to be limited to infection- and injury-driven carcinogenesis, where they are activated by pathogenic ligands. With the recognition of damage-associated molecular patterns (DAMPs) as ligands of PRRs, the role of PRRs in carcinogenesis has also been implicated in other non-pathogen-driven neoplasms. Dying (apoptotic or necrotic) cells shed a plethora of DAMPs causing persistent activation of PRRs, leading to chronic inflammation and carcinogenesis. Such chronic activation of TLRs promotes tumor cell proliferation and enhances tumor cell invasion and metastasis by regulating pro-inflammatory cytokines, metalloproteinases, and integrins. Due to the decisive role of PRRs in carcinogenesis, targeting PRRs appears to be an effective cancer-preventive strategy. This review provides a brief account on the association of PRRs with various cancers and their role in carcinogenesis. PMID:26279628

  14. Molecular Recognition of Insulin by a Synthetic Receptor

    SciTech Connect

    Chinai, Jordan M.; Taylor, Alexander B.; Ryno, Lisa M.; Hargreaves, Nicholas D.; Morris, Christopher A.; Hart, P. John; Urbach, Adam R.

    2011-08-29

    The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 x 10{sup 6} M{sup -1} and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7{center_dot}insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

  15. Selective recognition of tetrahedral dianions by a hexaaza cryptand receptor.

    PubMed

    Mateus, Pedro; Delgado, Rita; Brandão, Paula; Carvalho, Sílvia; Félix, Vítor

    2009-11-21

    A hexaamine cage was synthesised in good yield by a [2+3] Schiff-base condensation followed by sodium borohydride reduction to be used as a receptor for the selective binding of anionic species. The protonation constants of the receptor, as well as its association constants with Cl(-), I(-), NO(3)(-), AcO(-), ClO(4)(-), H(2)PO(4)(-), SO(4)(2-), SeO(4)(2-) and S(2)O(3)(2-) were determined by potentiometry at 298.2 +/- 0.1 K in H(2)O-MeOH (50 : 50 v/v) and at ionic strength 0.10 +/- 0.01 mol dm(-3) in KTsO. These studies revealed a remarkable selectivity for dianionic tetrahedral anions by the protonated receptor, with association constants ranging 5.03-5.30 log units for the dianionic species and 1.49-2.97 log units for monoanionic ones. Single crystal X-ray determination of [(H(6)xyl)(SO(4))(H(2)O)(6)](SO(4))(2).9.5H(2)O showed that one sulfate anion is encapsulated into the receptor cage sited between the two 2,4,6-triethylbenzene caps establishing three N-HO hydrogen bonds with two adjacent N-H binding sites and additional O-HO hydrogen bonding interactions with six water of crystallization molecules. Four water molecules of the (SO(4))(H(2)O)(6) cluster interact with [H(6)xyl](6+) through N-HO hydrogen bonds. Molecular dynamics simulations (MD) carried out with SO(4)(2-) and Cl(-) anions in H(2)O-MeOH (50 : 50 v/v) allowed the full understanding of anion molecular recognition, the selectivity of the protonated receptor for SO(4)(2-) and the role played by the methanol and water solvent molecules. PMID:19865702

  16. Recognition of Human Oncogenic Viruses by Host Pattern-Recognition Receptors

    PubMed Central

    Di Paolo, Nelson C.

    2014-01-01

    Human oncogenic viruses include Epstein–Barr virus, hepatitis B virus, hepatitis C virus, human papilloma virus, human T-cell lymphotropic virus, Kaposi’s associated sarcoma virus, and Merkel cell polyomavirus. It would be expected that during virus–host interaction, the immune system would recognize these pathogens and eliminate them. However, through evolution, these viruses have developed a number of strategies to avoid such an outcome and successfully establish chronic infections. The persistent nature of the infection caused by these viruses is associated with their oncogenic potential. In this article, we will review the latest information on the interaction between oncogenic viruses and the innate immune system of the host. In particular, we will summarize the available knowledge on the recognition by host pattern-recognition receptors of pathogen-associated molecular patterns present in the incoming viral particle or generated during the virus’ life cycle. We will also review the data on the recognition of cell-derived danger associated molecular patterns generated during the virus infection that may impact the outcome of the host–pathogen interaction and the development cancer. PMID:25101093

  17. Recognition of human oncogenic viruses by host pattern-recognition receptors.

    PubMed

    Di Paolo, Nelson C

    2014-01-01

    Human oncogenic viruses include Epstein-Barr virus, hepatitis B virus, hepatitis C virus, human papilloma virus, human T-cell lymphotropic virus, Kaposi's associated sarcoma virus, and Merkel cell polyomavirus. It would be expected that during virus-host interaction, the immune system would recognize these pathogens and eliminate them. However, through evolution, these viruses have developed a number of strategies to avoid such an outcome and successfully establish chronic infections. The persistent nature of the infection caused by these viruses is associated with their oncogenic potential. In this article, we will review the latest information on the interaction between oncogenic viruses and the innate immune system of the host. In particular, we will summarize the available knowledge on the recognition by host pattern-recognition receptors of pathogen-associated molecular patterns present in the incoming viral particle or generated during the virus' life cycle. We will also review the data on the recognition of cell-derived danger associated molecular patterns generated during the virus infection that may impact the outcome of the host-pathogen interaction and the development cancer.

  18. The Role of Pattern Recognition Receptors in Intestinal Inflammation

    PubMed Central

    Fukata, Masayuki; Arditi, Moshe

    2013-01-01

    Recognition of microorganisms by pattern recognition receptors (PRRs) is the primary component of innate immunity that is responsible for the maintenance of host-microbial interactions in intestinal mucosa. Disregulation in host-commensal interactions has been implicated as the central pathogenesis of inflammatory bowel disease (IBD), which predisposes to developing colorectal cancer. Recent animal studies have begun to outline some unique physiology and pathology involving each PRR signaling in the intestine. The major roles played by PRRs in the gut appear to be regulation of the number and the composition of commensal bacteria, epithelial proliferation and mucosal permiability in response to epithelial injury. In addition, PRR signaling in lamina propria immune cells may be involved in induction of inflammation in response to invasion of pathogens. Because some PRR-deficient mice have shown variable susceptibility to colitis, the outcome of intestinal inflammation may be modified depending on PRR signaling in epithelial cells, immune cells, and the composition of commensal flora. Through recent findings in animal models of IBD, this review will discuss how abnormal PRR signaling may contribute to the pathogenesis of inflammation and inflammation-associated tumorigenesis in the intestine. PMID:23515136

  19. Autophagy and Pattern Recognition Receptors in Innate Immunity

    PubMed Central

    Delgado, Monica; Singh, Sudha; De Haro, Sergio; Master, Sharon; Ponpuak, Marisa; Dinkins, Christina; Ornatowski, Wojchiech; Vergne, Isabelle; Deretic, Vojo

    2009-01-01

    Summary Autophagy is a physiologically and immunologically controlled intracellular homeostatic pathway that sequesters and degrades cytoplasmic targets including macromolecular aggregates, cellular organelles such as mitochondria, and whole microbes or their products. Recent advances show that autophagy plays a role in innate immunity in several ways: (i) direct elimination of intracellular microbes by digestion in autolysosomes, (ii) delivery of cytosolic microbial products to pattern recognition receptors (PRRs) in a process referred to as topological inversion, and (iii) as an antimicrobial effector of Toll-like receptors and other PRR signaling. Autophagy eliminates pathogens in vitro and in vivo but, when aberrant due to mutations, contributes to human inflammatory disorders such as Crohn's disease. In this review, we examine these relationships and propose that autophagy is one of the most ancient innate immune defenses that has possibly evolved at the time of α-protobacteria-pre-eukaryote relationships, leading up to modern eukaryotic cell-mitochondrial symbiosis, and that during the metazoan evolution, additional layers of immunological regulation have been superimposed and integrated with this primordial innate immunity mechanism. PMID:19120485

  20. BSA-imprinted synthetic receptor for reversible template recognition.

    PubMed

    Wang, Huafang; He, Yunhua; He, Xiwen; Li, Wenyou; Chen, Langxing; Zhang, Yukui

    2009-06-01

    A novel approach to the manufacturing of protein-responsive imprints on a home-made chitosan substrate was established together with m-aminophenylboronic acid (APBA) as a functional monomer. The produced polymers were characterized using both (1) equilibrium adsorption assays and (2) high performance liquid chromatography analysis. Results confirmed that the synthesized BSA-MIP (molecularly imprinted polymer) has a high affinity towards its template compared to the determined control proteins. The produced BSA-MIP featured largely in its good adsorption reversibility, especially in competitive binding assays, which is of great biological significance in separations. Non-specific binding was reduced to almost zero in a BSA/BHb competitive binding event. An excellent HPLC profile of template recognition was found for BSA-MIP, even under harsh mobile phase conditions. In the present work, the adopted trapped-template-release method permits recovery of bound BSA [1]. The strategy of making an artificial protein-receptor with high adsorption affinity and reversibility is promising in on-line isolation of target protein from complicated biological environments.

  1. Pattern recognition receptors as potential therapeutic targets in inflammatory rheumatic disease.

    PubMed

    Mullen, Lisa M; Chamberlain, Giselle; Sacre, Sandra

    2015-05-15

    The pattern recognition receptors of the innate immune system are part of the first line of defence against pathogens. However, they also have the ability to respond to danger signals that are frequently elevated during tissue damage and at sites of inflammation. Inadvertent activation of pattern recognition receptors has been proposed to contribute to the pathogenesis of many conditions including inflammatory rheumatic diseases. Prolonged inflammation most often results in pain and damage to tissues. In particular, the Toll-like receptors and nucleotide-binding oligomerisation domain-like receptors that form inflammasomes have been postulated as key contributors to the inflammation observed in rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus. As such, there is increasing interest in targeting these receptors for therapeutic treatment in the clinic. Here the role of pattern recognition receptors in the pathogenesis of these diseases is discussed, with an update on the development of interventions to modulate the activity of these potential therapeutic targets.

  2. Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases.

    PubMed

    Mbengue, Malick; Bourdais, Gildas; Gervasi, Fabio; Beck, Martina; Zhou, Ji; Spallek, Thomas; Bartels, Sebastian; Boller, Thomas; Ueda, Takashi; Kuhn, Hannah; Robatzek, Silke

    2016-09-27

    Sensing of potential pathogenic bacteria is of critical importance for immunity. In plants, this involves plasma membrane-resident pattern recognition receptors, one of which is the FLAGELLIN SENSING 2 (FLS2) receptor kinase. Ligand-activated FLS2 receptors are internalized into endosomes. However, the extent to which these spatiotemporal dynamics are generally present among pattern recognition receptors (PRRs) and their regulation remain elusive. Using live-cell imaging, we show that at least three other receptor kinases associated with plant immunity, PEP RECEPTOR 1/2 (PEPR1/2) and EF-TU RECEPTOR (EFR), internalize in a ligand-specific manner. In all cases, endocytosis requires the coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1), and thus depends on receptor activation status. We also show the internalization of liganded FLS2, suggesting the transport of signaling competent receptors. Trafficking of activated PRRs requires clathrin and converges onto the same endosomal vesicles that are also shared with the hormone receptor BRASSINOSTERIOD INSENSITIVE 1 (BRI1). Importantly, clathrin-dependent endocytosis participates in plant defense against bacterial infection involving FLS2-mediated stomatal closure and callose deposition, but is uncoupled from activation of the flagellin-induced oxidative burst and MAP kinase signaling. In conclusion, immunity mediated by pattern recognition receptors depends on clathrin, a critical component for the endocytosis of signaling competent receptors into a common endosomal pathway. PMID:27651493

  3. Oxytocin, vasopressin and estrogen receptor gene expression in relation to social recognition in female mice

    PubMed Central

    Clipperton-Allen, Amy E.; Lee, Anna W.; Reyes, Anny; Devidze, Nino; Phan, Anna; Pfaff, Donald W.; Choleris, Elena

    2012-01-01

    Inter- and intra-species differences in social behavior and recognition-related hormones and receptors suggest that different distribution and/or expression patterns may relate to social recognition. We used qRT-PCR to investigate naturally occurring differences in expression of estrogen receptor-alpha (ERα), ER-beta (ERβ), progesterone receptor (PR), oxytocin (OT) and receptor, and vasopressin (AVP) and receptors in proestrous female mice. Following four 5 min exposures to the same two conspecifics, one was replaced with a novel mouse in the final trial (T5). Gene expression was examined in mice showing high (85–100%) and low (40–60%) social recognition scores (i.e., preferential novel mouse investigation in T5) in eight socially-relevant brain regions. Results supported OT and AVP involvement in social recognition, and suggest that in the medial preoptic area, increased OT and AVP mRNA, together with ERα and ERβ gene activation, relate to improved social recognition. Initial social investigation correlated with ERs, PR and OTR in the dorsolateral septum, suggesting that these receptors may modulate social interest without affecting social recognition. Finally, increased lateral amygdala gene activation in the LR mice may be associated with general learning impairments, while decreased lateral amygdala activity may indicate more efficient cognitive mechanisms in the HR mice. PMID:22079582

  4. Molecular Recognition of Paired Receptors in the Immune System

    PubMed Central

    Kuroki, Kimiko; Furukawa, Atsushi; Maenaka, Katsumi

    2012-01-01

    Cell surface receptors are responsible for regulating cellular function on the front line, the cell membrane. Interestingly, accumulating evidence clearly reveals that the members of cell surface receptor families have very similar extracellular ligand-binding regions but opposite signaling systems, either inhibitory or stimulatory. These receptors are designated as paired receptors. Paired receptors often recognize not only physiological ligands but also non-self ligands, such as viral and bacterial products, to fight infections. In this review, we introduce several representative examples of paired receptors, focusing on two major structural superfamilies, the immunoglobulin-like and the C-type lectin-like receptors, and explain how these receptors distinguish self and non-self ligands to maintain homeostasis in the immune system. We further discuss the evolutionary aspects of these receptors as well as the potential drug targets for regulating diseases. PMID:23293633

  5. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns (PAMPs) and endogenous molecules...

  6. Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer.

    PubMed

    Šunderić, Miloš; Šedivá, Alena; Robajac, Dragana; Miljuš, Goran; Gemeiner, Peter; Nedić, Olgica; Katrlík, Jaroslav

    2016-07-01

    Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.

  7. Human plasma alpha 2-macroglobulin promotes in vitro oxidative stress cracking of Pellethane 2363-80A: in vivo and in vitro correlations.

    PubMed

    Zhao, Q H; McNally, A K; Rubin, K R; Renier, M; Wu, Y; Rose-Caprara, V; Anderson, J M; Hiltner, A; Urbanski, P; Stokes, K

    1993-03-01

    It is hypothesized in this study that the phenomenon of environmental stress cracking (ESC) in polyetherurethane is caused by a synergistic action of biological components in the body fluids, oxidative agents, and stress. An in vitro system is designed to mimic the in vivo system; human plasma contains certain biological components that can act as a stress cracking promoter, while H2O2 (Co) solution provides an oxidative reaction comparable to that observed in the respiratory burst of adherent macrophages and foreign-body giant cells. It is demonstrated that the phenomenon of in vivo stress cracking in Pellethane 2363-80A is duplicated by an in vitro system that involves a pretreatment of prestressed specimens with human plasma at 37 degrees C for 7 days followed by oxidation in 10% hydrogen peroxide with 0.10M cobalt chloride at 50 degrees C for 10 days. The pretreatment with plasma has a synergistic effect with the oxidation by H2O2 (Co) treatment to produce ESC. A plasma component responsible for promoting stress cracking in Pellethane polyurethane is identified to be alpha 2-macroglobulin (alpha 2M). PMID:7689567

  8. Human sputum cathepsin B degrades proteoglycan, is inhibited by alpha 2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C.

    PubMed Central

    Buttle, D J; Abrahamson, M; Burnett, D; Mort, J S; Barrett, A J; Dando, P M; Hill, S L

    1991-01-01

    The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation. Images Fig. 3. Fig. 4. Fig. 5. PMID:1710889

  9. Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Ma, Yuyuan; Lv, Maomin; Jia, Junting; Zhao, Xiong; Zhang, Jingang

    2016-07-01

    As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource. PMID:27214605

  10. Molecular recognition of organic ammonium ions in solution using synthetic receptors

    PubMed Central

    Späth, Andreas

    2010-01-01

    Summary Ammonium ions are ubiquitous in chemistry and molecular biology. Considerable efforts have been undertaken to develop synthetic receptors for their selective molecular recognition. The type of host compounds for organic ammonium ion binding span a wide range from crown ethers to calixarenes to metal complexes. Typical intermolecular interactions are hydrogen bonds, electrostatic and cation–π interactions, hydrophobic interactions or reversible covalent bond formation. In this review we discuss the different classes of synthetic receptors for organic ammonium ion recognition and illustrate the scope and limitations of each class with selected examples from the recent literature. The molecular recognition of ammonium ions in amino acids is included and the enantioselective binding of chiral ammonium ions by synthetic receptors is also covered. In our conclusion we compare the strengths and weaknesses of the different types of ammonium ion receptors which may help to select the best approach for specific applications. PMID:20502608

  11. Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor.

    PubMed

    Zamze, Susanne; Martinez-Pomares, Luisa; Jones, Hannah; Taylor, Philip R; Stillion, Richard J; Gordon, Siamon; Wong, Simon Y C

    2002-11-01

    The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.

  12. Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor.

    PubMed

    Zamze, Susanne; Martinez-Pomares, Luisa; Jones, Hannah; Taylor, Philip R; Stillion, Richard J; Gordon, Siamon; Wong, Simon Y C

    2002-11-01

    The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed. PMID:12196537

  13. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    SciTech Connect

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A.

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  14. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    PubMed Central

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Charles L.; Cooper, Max D.; Wilson, Ian A.

    2012-01-01

    Summary Variable Lymphocyte Receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin-fold based T- and B-cell receptors, lymphocyte-like cells of jawless fish express VLRs (A, B or C) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA, C) in function. Here we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores. PMID:22405006

  15. Pattern Recognition Receptors and Cytokines in Mycobacterium tuberculosis Infection—The Double-Edged Sword?

    PubMed Central

    Hossain, Md. Murad; Norazmi, Mohd-Nor

    2013-01-01

    Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), remains a major cause of human death worldwide. Innate immunity provides host defense against Mtb. Phagocytosis, characterized by recognition of Mtb by macrophages and dendritic cells (DCs), is the first step of the innate immune defense mechanism. The recognition of Mtb is mediated by pattern recognition receptors (PRRs), expressed on innate immune cells, including toll-like receptors (TLRs), complement receptors, nucleotide oligomerization domain like receptors, dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin (DC-SIGN), mannose receptors, CD14 receptors, scavenger receptors, and FCγ receptors. Interaction of mycobacterial ligands with PRRs leads macrophages and DCs to secrete selected cytokines, which in turn induce interferon-γ- (IFNγ-) dominated immunity. IFNγ and other cytokines like tumor necrosis factor-α (TNFα) regulate mycobacterial growth, granuloma formation, and initiation of the adaptive immune response to Mtb and finally provide protection to the host. However, Mtb can evade destruction by antimicrobial defense mechanisms of the innate immune system as some components of the system may promote survival of the bacteria in these cells and facilitate pathogenesis. Thus, although innate immunity components generally play a protective role against Mtb, they may also facilitate Mtb survival. The involvement of selected PRRs and cytokines on these seemingly contradictory roles is discussed. PMID:24350246

  16. Biochemical study of multiple drug recognition sites on central benzodiazepine receptors

    SciTech Connect

    Trifiletti, R.R.

    1986-01-01

    The benzodiazepine receptor complex of mammalian brain possesses recognition sites which mediate (at least in part) the pharmacologic actions of the 1,4-benzodiazepines and barbiturates. Evidence is provided suggesting the existence of least seven distinct drug recognition sites on this complex. Interactions between the various recognition sites have been explored using radioligand binding techniques. This information is utilized to provide a comprehensive scheme for characterizing receptor-active drugs on an anxiolytic-anticonvulsant/proconvulsant continuum using radioligand binding techniques, as well as a comprehensive program for identifying potential endogenous receptor-active substances. Further evidence is provided here supporting the notion of benzodiazepine recognition site heterogeneity. Classical 1,4-benzodiazepines do not appear to differentiate two populations of benzodiazepine receptors in an equilibrium sense, but appear to do so in a kinetic sense. An apparent physical separation of the two receptor subtypes can be achieved by differential solubilization. The benzodiazepine binding subunit can be identified by photoaffinity labeling with the benzodiazepine agonist (/sup 3/H)flunitrazepan. Conditions for reproducible partial proteolytic mapping of (/sup 3/H)flunitrazepam photoaffinity labeled receptors are established. From these maps, it is concluded that there are probably no major differences in the primary sequence of the benzodiazepine binding subunit in various regions of the rat central nervous system.

  17. Distinctive Recognition of Flagellin by Human and Mouse Toll-Like Receptor 5

    PubMed Central

    Forstnerič, Vida; Ivičak-Kocjan, Karolina; Ljubetič, Ajasja; Jerala, Roman; Benčina, Mojca

    2016-01-01

    Toll-like receptor 5 (TLR5) is a receptor of the innate immune system that recognizes flagellin from certain bacterial species and triggers an inflammatory response. The Salmonella dublin flagellin in complex with zebrafish TLR5 has been crystallized previously. In the present study, we extrapolate the structure of this complex using structure-guided mutagenesis to determine the recognition modes of human and mouse TLR5 receptors and demonstrate species-specific differences in flagellin recognition. In general, the recognition mode of the mouse receptor can be said to be more robust in comparison to that of the human receptor. All-atom molecular dynamics simulation showed differences between the two receptors within the primary binding region. Using a functional motility assay, we show that although the highly conserved area of the flagellin analyzed in this study encompasses key structural requirements for flagella formation, a direct correlation between immune recognition and structure on the level of amino acid residues is not observed. PMID:27391968

  18. Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

    PubMed

    Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R; Jiang, Haobo

    2015-07-01

    Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. PMID:25701384

  19. Genetic variation in pattern recognition receptors: functional consequences and susceptibility to infectious disease.

    PubMed

    Jaeger, Martin; Stappers, Mark H T; Joosten, Leo A B; Gyssens, Inge C; Netea, Mihai G

    2015-01-01

    Cells of the innate immune system are equipped with surface and cytoplasmic receptors for microorganisms called pattern recognition receptors (PRRs). PRRs recognize specific pathogen-associated molecular patterns and as such are crucial for the activation of the immune system. Currently, five different classes of PRRs have been described: Toll-like receptors, C-type lectin receptors, nucleotide-binding oligomerization domain-like receptors, retinoic acid-inducible gene I-like receptors and absent in melanoma 2-like receptors. Following their discovery, many sequence variants in PRR genes have been uncovered and shown to be implicated in human infectious diseases. In this review, we will discuss the effect of genetic variation in PRRs and their signaling pathways on susceptibility to infectious diseases in humans.

  20. Recognition of Legionella pneumophila nucleic acids by innate immune receptors.

    PubMed

    Cunha, Larissa D; Zamboni, Dario S

    2014-12-01

    Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection.

  1. Analysis of serum β-amyloid peptides, α2-macroglobulin, complement factor H, and clusterin levels in APP/PS1 transgenic mice during progression of Alzheimer's disease.

    PubMed

    Wang, Dejiang; Di, Xiangjun; Fu, Lu; Li, Yingnan; Han, Xiao; Wu, Hui; Cai, Linjun; Meng, Xiangyu; Jiang, Chunlai; Kong, Wei; Su, Weiheng

    2016-10-19

    As a progressive age-related neurodegenerative disorder, Alzheimer's disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of β-amyloid proteins, α2-macroglobulin (α-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum Aβ40, Aβ42, α-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum Aβ42 levels and Aβ42/Aβ40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The α-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum Aβ42 level, Aβ42/Aβ40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD. PMID:27541273

  2. Sugar recognition: designing artificial receptors for applications in biological diagnostics and imaging.

    PubMed

    Miron, Caitlin E; Petitjean, Anne

    2015-02-01

    At the cellular level, numerous processes ranging from protein folding to disease development are mediated by a sugar-based molecular information system that is much less well known than its DNA- or protein-based counterparts. The subtle structural diversity of such sugar tags nevertheless offers an excellent, if challenging, opportunity to design receptors for the selective recognition of biorelevant sugars. Over the past 40 years, growing interest in the field of sugar recognition has led to the development of several promising artificial receptors, which could soon find widespread use in medical diagnostics and cell imaging.

  3. Gels based on anion recognition between triurea receptor and phosphate anion.

    PubMed

    Yang, Cuiling; Wu, Biao; Chen, Yongming; Zhang, Ke

    2015-04-01

    Anion recognition between the triurea receptor and phosphate anion is demonstrated as the cross-linkage to build supramolecular polymer gels for the first time. A novel multi-block copolymer (3) is designed to have functional triurea groups as cross-linking units along the polymer main chain. By virtue of anion coordination between the triurea receptor and phosphate anion with a binding mode of 2:1, supramolecular polymer gels are then prepared based on anion recognition using 3 as the building block. PMID:25694389

  4. [Probable mechanism of recognition of cholinergic ligands by acetylcholine receptors].

    PubMed

    Demushkin, V P; Kotelevtsev, Iu V; Pliashkevich, Iu G; Khramtsov, N V

    1982-01-01

    Dryding's models were used for the conformational analysis of compounds affecting muscarin-specific acetylcholine receptor and nicotin-specific acetylcholine receptor. Ammonium group and ether oxygen (3.6 A apart from the ammonium group) specifically oriented to each other were shown to be necessary structural elements to reveal muscarin-type cholinergic activity. Ammonium group along with carbonyl oxygen or its substituent (5 A distance) are the necessary structural units providing nicotin-type cholinergic activity. The presence of two hydrophobic substituents (one in the ammonium area and the other neighbouring the second active grouping) is the additional factor. The developed principles were justified by the use of a series of synthetic samples. The compounds were obtained likely favouring affinitive modification of acetylcholine receptor (dissociation constants of acetylcholine receptor complexes equalling to 10(-4)--10(-7) M-1). PMID:7070378

  5. Structural Basis for Molecular Recognition at Serotonin Receptors

    PubMed Central

    Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D.; Gao, Xiang; Zhou, Edward X.; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.; Xu, H. Eric

    2013-01-01

    Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist anti-migraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared to the accompanying structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs. PMID:23519210

  6. Identification of a novel V1-type AVP receptor based on the molecular recognition theory.

    PubMed Central

    Herrera, V. L.; Ruiz-Opazo, N.

    2001-01-01

    BACKGROUND: The molecular recognition theory predicts that binding domains of peptide hormones and their corresponding receptor binding domains evolved from complementary strands of genomic DNA, and that a process of selective evolutionary mutational events within these primordial domains gave rise to the high affinity and high specificity of peptide hormone-receptor interactions observed today in different peptide hormone-receptor systems. Moreover, this theory has been broadened as a general hypothesis that could explain the evolution of intermolecular protein-protein and intramolecular peptide interactions. MATERIALS AND METHODS: Applying a molecular cloning strategy based on the molecular recognition theory, we screened a rat kidney cDNA library with a vasopressin (AVP) antisense oligonucleotide probe, expecting to isolate potential AVP receptors. RESULTS: We isolated a rat kidney cDNA encoding a functional V1-type vasopressin receptor. Structural analysis identified a 135 amino acid-long polypeptide with a single transmembrane domain, quite distinct from the rhodopsin-based G protein-coupled receptor superfamily. Functional analysis of the expressed V1-type receptor in Cos-1 cells revealed AVP-specific binding, AVP-specific coupling to Ca2+ mobilizing transduction system, and characteristic V1-type antagonist inhibition. CONCLUSIONS: This is the second AVP receptor cDNA isolated using AVP antipeptide-based oligonucleotide screening, thus providing compelling evidence in support of the molecular recognition theory as the basis of the evolution of this peptide hormone-receptor system, as well as adds molecular complexity and diversity to AVP receptor systems. PMID:11683375

  7. A tricatecholic receptor for carbohydrate recognition: synthesis and binding studies.

    PubMed

    Cacciarini, Martina; Cordiano, Elisa; Nativi, Cristina; Roelens, Stefano

    2007-05-11

    A new tripodal receptor bearing three catechol subunits on a benzene platform has been synthesized in four steps from 1,3,5-triethylbenzene and pyrogallol. The binding ability of the tricatecholic receptor was investigated toward several monosaccharides in CDCl3, where multiple equilibria were detected, and compared to that of a previously reported trisureidic receptor of analogous structure. Association constants were measured by 1H NMR titrations, and the corresponding affinities were assessed through the BC50 parameter, a binding descriptor univocally defining the affinity of a host for a guest in multi-equilibrium systems. Results show that the tripodal catecholic receptor binds the octyl glycosides with affinities ranging from 0.87 to 5.2 mM and with a 6-fold selectivity factor for the alpha-mannoside over the beta-glucoside. Although the affinity for glycosides was not appreciably improved with respect to the ureidic receptor, a significant change in selectivity was obtained by the H-bonding group replacement.

  8. Oxoanion Recognition by Benzene-based Tripodal Pyrrolic Receptors

    SciTech Connect

    Bill, Nathan; Kim, Dae-Sik; Kim, Sung Kuk; Park, Jung Su; Lynch, Vincent M.; Young, Neil J; Hay, Benjamin; Yang, Youjun; Anslyn, Eric; Sessler, Jonathan L.

    2012-01-01

    Two new tripodal receptors based on pyrrole- and dipyrromethane-functionalised derivatives of a sterically geared precursor, 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene, are reported; these systems, compounds 1 and 2, display high affinity and selectivity for tetrahedral anionic guests, in particular dihydrogen phosphate, pyrophosphate and hydrogen sulphate, in acetonitrile as inferred from isothermal titration calorimetry measurements. Support for the anion-binding ability of these systems comes from theoretical calculations and a single-crystal X-ray diffraction structure of the 2:2 (host:guest) dihydrogen phosphate complex is obtained in the case of the pyrrole-based receptor system, 1. Keywords anion receptors, dihydrogen phosphate, hydrogen sulphate, X-ray structure, theoretical calculations.

  9. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  10. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

  11. Interaction of human tissue plasminogen activator (t-PA) with pregnancy zone protein: a comparative study with t-PA-alpha2-macroglobulin interaction.

    PubMed

    Sánchez, M C; Chiabrando, G A; Guglielmone, H A; Bonacci, G R; Rabinovich, G A; Vides, M A

    1998-08-01

    Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

  12. The interleukin 1 alpha, interleukin 1 beta, interleukin 6 and alpha-2-macroglobulin serum levels in patients with early or late onset Alzheimer's disease, mild cognitive impairment or Parkinson's disease.

    PubMed

    Dursun, Erdinç; Gezen-Ak, Duygu; Hanağası, Haşmet; Bilgiç, Başar; Lohmann, Ebba; Ertan, Sibel; Atasoy, İrem L; Alaylıoğlu, Merve; Araz, Ömür Selin; Önal, Burak; Gündüz, Ayşegül; Apaydın, Hülya; Kızıltan, Güneş; Ulutin, Turgut; Gürvit, Hakan; Yılmazer, Selma

    2015-06-15

    Alzheimer's disease (EOAD, LOAD), mild cognitive impairment (MCI), Parkinson's disease (PD) and healthy controls were included to determine the serum interleukin-1s (IL-1α, IL-1β), IL-6 and alpha-2-macroglobulin (α2M) levels using ELISA. IL-6 might be a significant contributor to the inflammatory response in LOAD. The MCI data indicate that IL-1s, α2M and BDNF are somehow related, and this relationship might allow MCI patients to be more similar to the healthy controls. A correlation analysis of multiple biomarkers in different neurodegenerative disorders might be more useful than determining the levels of a single cytokine in a single disorder.

  13. E3 Ubiquitin Ligases Pellinos as Regulators of Pattern Recognition Receptor Signaling and Immune responses

    PubMed Central

    Medvedev, Andrei E.; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-01-01

    SUMMARY Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an IL-1 receptor-associated kinase homologue in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2 and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g., Pellino-1 being a negative regulator in T-lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we have summarized current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and TNF receptors, and discuss Pellino’s role in sepsis and infectious diseases, as well as in autoimmune, inflammatory and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies. PMID:26085210

  14. E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses.

    PubMed

    Medvedev, Andrei E; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-07-01

    Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.

  15. Role of Toll-Like Receptor 13 in Innate Immune Recognition of Group B Streptococci

    PubMed Central

    Signorino, Giacomo; Mohammadi, Nastaran; Patanè, Francesco; Buscetta, Marco; Venza, Mario; Venza, Isabella; Mancuso, Giuseppe; Midiri, Angelina; Alexopoulou, Lena; Teti, Giuseppe; Beninati, Concetta

    2014-01-01

    Murine Toll-like receptor 13 (TLR13), an endosomal receptor that is not present in humans, is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA (23S rRNA). Little is known, however, of the impact of TLR13 on antibacterial host defenses. Here we examined the role of this receptor in the context of infection induced by the model pathogen group B streptococcus (GBS). To this end, we used bacterial strains masked from TLR13 recognition by virtue of constitutive expression of the ErmC methyltransferase, which results in dimethylation of the 23S rRNA motif at a critical adenine residue. We found that TLR13-mediated rRNA recognition was required for optimal induction of tumor necrosis factor alpha and nitrous oxide in dendritic cell and macrophage cultures stimulated with heat-killed bacteria or purified bacterial RNA. However, TLR13-dependent recognition was redundant when live bacteria were used as a stimulus. Moreover, masking bacterial rRNA from TLR13 recognition did not increase the ability of GBS to avoid host defenses and replicate in vivo. In contrast, increased susceptibility to infection was observed under conditions in which signaling by all endosomal TLRs was abolished, i.e., in mice with a loss-of-function mutation in the chaperone protein UNC93B1. Our data lend support to the conclusion that TLR13 participates in GBS recognition, although blockade of the function of this receptor can be compensated for by other endosomal TLRs. Lack of selective pressure by bacterial infections might explain the evolutionary loss of TLR13 in humans. However, further studies using different bacterial species are needed to prove this hypothesis. PMID:25225249

  16. Chiral diaminopyrrolic receptors for selective recognition of mannosides, part 1: design, synthesis, and affinities of second-generation tripodal receptors.

    PubMed

    Nativi, Cristina; Francesconi, Oscar; Gabrielli, Gabriele; Vacca, Alberto; Roelens, Stefano

    2011-04-18

    A new generation of chiral tripodal receptors for recognition of carbohydrates, featuring trans-1,2-diaminocyclohexane as a key structural element, and their recognition properties toward a set of glycosides of biologically relevant monosaccharides is described. The introduction of a chelating diamino unit into the pyrrolic tripodal architecture markedly enhanced their binding abilities compared with the parent aminopyrrolic receptors previously reported by our group. In addition, the chirality of the structure had a clear impact on affinities, as well as on selectivities, displaying high enantiodiscrimination levels. These second-generation diaminopyrrolic tripodal receptors are highly selective for mannose among other monosaccharides, with two members of the family being selective for the α and the β anomers respectively. The measured affinities in acetonitrile, 83 μM of (S)-7 for the β mannoside and 127 μM of (R)-5 for the α mannoside, make them the most effective synthetic receptors for mannosides reported to date. The affinity assessment required a further evolution of the BC(0)(50) parameter, a previously developed binding descriptor, which in its ultimate formulation has now been extended to include, with no restrictions, complexes of any stoichiometry, and can thus be generally employed to rank affinity data from heterogeneous systems on a common scale.

  17. Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

    NASA Astrophysics Data System (ADS)

    Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

    Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

  18. Structural basis of transcobalamin recognition by human CD320 receptor

    PubMed Central

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  19. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-07-14

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  20. Structural basis of transcobalamin recognition by human CD320 receptor

    NASA Astrophysics Data System (ADS)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  1. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  2. Non-classical amine recognition evolved in a large clade of olfactory receptors

    PubMed Central

    Li, Qian; Tachie-Baffour, Yaw; Liu, Zhikai; Baldwin, Maude W; Kruse, Andrew C; Liberles, Stephen D

    2015-01-01

    Biogenic amines are important signaling molecules, and the structural basis for their recognition by G Protein-Coupled Receptors (GPCRs) is well understood. Amines are also potent odors, with some activating olfactory trace amine-associated receptors (TAARs). Here, we report that teleost TAARs evolved a new way to recognize amines in a non-classical orientation. Chemical screens de-orphaned eleven zebrafish TAARs, with agonists including serotonin, histamine, tryptamine, 2-phenylethylamine, putrescine, and agmatine. Receptors from different clades contact ligands through aspartates on transmembrane α-helices III (canonical Asp3.32) or V (non-canonical Asp5.42), and diamine receptors contain both aspartates. Non-classical monoamine recognition evolved in two steps: an ancestral TAAR acquired Asp5.42, gaining diamine sensitivity, and subsequently lost Asp3.32. Through this transformation, the fish olfactory system dramatically expanded its capacity to detect amines, ecologically significant aquatic odors. The evolution of a second, alternative solution for amine detection by olfactory receptors highlights the tremendous structural versatility intrinsic to GPCRs. DOI: http://dx.doi.org/10.7554/eLife.10441.001 PMID:26519734

  3. Serotonin 2a Receptor and Serotonin 1a Receptor Interact Within the Medial Prefrontal Cortex During Recognition Memory in Mice

    PubMed Central

    Morici, Juan F.; Ciccia, Lucia; Malleret, Gaël; Gingrich, Jay A.; Bekinschtein, Pedro; Weisstaub, Noelia V.

    2015-01-01

    Episodic memory, can be defined as the memory for unique events. The serotonergic system one of the main neuromodulatory systems in the brain appears to play a role in it. The serotonin 2a receptor (5-HT2aR) one of the principal post-synaptic receptors for 5-HT in the brain, is involved in neuropsychiatric and neurological disorders associated with memory deficits. Recognition memory can be defined as the ability to recognize if a particular event or item was previously encountered and is thus considered, under certain conditions, a form of episodic memory. As human data suggest that a constitutively decrease of 5-HT2A signaling might affect episodic memory performance we decided to compare the performance of mice with disrupted 5-HT2aR signaling (htr2a−/−) with wild type (htr2a+/+) littermates in different recognition memory and working memory tasks that differed in the level of proactive interference. We found that ablation of 5-HT2aR signaling throughout development produces a deficit in tasks that cannot be solved by single item strategy suggesting that 5-HT2aR signaling is involved in interference resolution. We also found that in the absence of 5-HT2aR signaling serotonin has a deleterious effect on recognition memory retrieval through the activation of 5-HT1aR in the medial prefrontal cortex. PMID:26779016

  4. Serotonin 2a Receptor and Serotonin 1a Receptor Interact Within the Medial Prefrontal Cortex During Recognition Memory in Mice.

    PubMed

    Morici, Juan F; Ciccia, Lucia; Malleret, Gaël; Gingrich, Jay A; Bekinschtein, Pedro; Weisstaub, Noelia V

    2015-01-01

    Episodic memory, can be defined as the memory for unique events. The serotonergic system one of the main neuromodulatory systems in the brain appears to play a role in it. The serotonin 2a receptor (5-HT2aR) one of the principal post-synaptic receptors for 5-HT in the brain, is involved in neuropsychiatric and neurological disorders associated with memory deficits. Recognition memory can be defined as the ability to recognize if a particular event or item was previously encountered and is thus considered, under certain conditions, a form of episodic memory. As human data suggest that a constitutively decrease of 5-HT2A signaling might affect episodic memory performance we decided to compare the performance of mice with disrupted 5-HT2aR signaling (htr2a (-/-)) with wild type (htr2a (+/+)) littermates in different recognition memory and working memory tasks that differed in the level of proactive interference. We found that ablation of 5-HT2aR signaling throughout development produces a deficit in tasks that cannot be solved by single item strategy suggesting that 5-HT2aR signaling is involved in interference resolution. We also found that in the absence of 5-HT2aR signaling serotonin has a deleterious effect on recognition memory retrieval through the activation of 5-HT1aR in the medial prefrontal cortex. PMID:26779016

  5. International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

    PubMed Central

    Orr, Selinda; Ferguson, Brian; Symmons, Martyn F.; Boyle, Joseph P.; Monie, Tom P.

    2015-01-01

    Since the discovery of Toll, in the fruit fly Drosophila melanogaster, as the first described pattern recognition receptor (PRR) in 1996, many families of these receptors have been discovered and characterized. PRRs play critically important roles in pathogen recognition to initiate innate immune responses that ultimately link to the generation of adaptive immunity. Activation of PRRs leads to the induction of immune and inflammatory genes, including proinflammatory cytokines and chemokines. It is increasingly clear that many PRRs are linked to a range of inflammatory, infectious, immune, and chronic degenerative diseases. Several drugs to modulate PRR activity are already in clinical trials and many more are likely to appear in the near future. Here, we review the different families of mammalian PRRs, the ligands they recognize, the mechanisms of activation, their role in disease, and the potential of targeting these proteins to develop the anti-inflammatory therapeutics of the future. PMID:25829385

  6. International Union of Basic and Clinical Pharmacology. XCVI. Pattern recognition receptors in health and disease.

    PubMed

    Bryant, Clare E; Orr, Selinda; Ferguson, Brian; Symmons, Martyn F; Boyle, Joseph P; Monie, Tom P

    2015-01-01

    Since the discovery of Toll, in the fruit fly Drosophila melanogaster, as the first described pattern recognition receptor (PRR) in 1996, many families of these receptors have been discovered and characterized. PRRs play critically important roles in pathogen recognition to initiate innate immune responses that ultimately link to the generation of adaptive immunity. Activation of PRRs leads to the induction of immune and inflammatory genes, including proinflammatory cytokines and chemokines. It is increasingly clear that many PRRs are linked to a range of inflammatory, infectious, immune, and chronic degenerative diseases. Several drugs to modulate PRR activity are already in clinical trials and many more are likely to appear in the near future. Here, we review the different families of mammalian PRRs, the ligands they recognize, the mechanisms of activation, their role in disease, and the potential of targeting these proteins to develop the anti-inflammatory therapeutics of the future.

  7. On the logic of restrictive recognition of peptide by the T-cell antigen receptor

    PubMed Central

    2011-01-01

    This essay provides an analysis of the inadequacy of the current view of restrictive recognition of peptide by the T-cell antigen receptor. A competing model is developed, and the experimental evidence for the prevailing model is reinterpreted in the new framework. The goal is to contrast the two models with respect to their consistency, coverage of the data, explanatory power, and predictability. PMID:20931295

  8. Pyrrolic tripodal receptors for carbohydrates. Role of functional groups and binding geometry on carbohydrate recognition.

    PubMed

    Cacciarini, Martina; Nativi, Cristina; Norcini, Martina; Staderini, Samuele; Francesconi, Oscar; Roelens, Stefano

    2011-02-21

    The contribution from several H-bonding groups and the impact of geometric requirements on the binding ability of benzene-based tripodal receptors toward carbohydrates have been investigated by measuring the affinity of a set of structures toward octyl β-D-glucopyranoside, selected as a representative monosaccharide. The results reported in the present study demonstrate that a judicious choice of correct geometry and appropriate functional groups is critical to achieve the complementary hydrogen bonding interactions required for an effective carbohydrate recognition.

  9. NMDA Receptor Antagonist Ketamine Distorts Object Recognition by Reducing Feedback to Early Visual Cortex.

    PubMed

    van Loon, Anouk M; Fahrenfort, Johannes J; van der Velde, Bauke; Lirk, Philipp B; Vulink, Nienke C C; Hollmann, Markus W; Scholte, H Steven; Lamme, Victor A F

    2016-05-01

    It is a well-established fact that top-down processes influence neural representations in lower-level visual areas. Electrophysiological recordings in monkeys as well as theoretical models suggest that these top-down processes depend on NMDA receptor functioning. However, this underlying neural mechanism has not been tested in humans. We used fMRI multivoxel pattern analysis to compare the neural representations of ambiguous Mooney images before and after they were recognized with their unambiguous grayscale version. Additionally, we administered ketamine, an NMDA receptor antagonist, to interfere with this process. Our results demonstrate that after recognition, the pattern of brain activation elicited by a Mooney image is more similar to that of its easily recognizable grayscale version than to the pattern evoked by the identical Mooney image before recognition. Moreover, recognition of Mooney images decreased mean response; however, neural representations of separate images became more dissimilar. So from the neural perspective, unrecognizable Mooney images all "look the same", whereas recognized Mooneys look different. We observed these effects in posterior fusiform part of lateral occipital cortex and in early visual cortex. Ketamine distorted these effects of recognition, but in early visual cortex only. This suggests that top-down processes from higher- to lower-level visual areas might operate via an NMDA pathway. PMID:25662715

  10. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    PubMed

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  11. An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp

    PubMed Central

    Jang, Ji-Hyun; Shin, Hee Woong; Lee, Jung Min; Lee, Hyeon-Woo; Kim, Eun-Cheol; Park, Sang Hyuk

    2015-01-01

    Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation. PMID:26576076

  12. An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp.

    PubMed

    Jang, Ji-Hyun; Shin, Hee Woong; Lee, Jung Min; Lee, Hyeon-Woo; Kim, Eun-Cheol; Park, Sang Hyuk

    2015-01-01

    Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation.

  13. Structural insights into cargo recognition by the yeast PTS1 receptor.

    PubMed

    Hagen, Stefanie; Drepper, Friedel; Fischer, Sven; Fodor, Krisztian; Passon, Daniel; Platta, Harald W; Zenn, Michael; Schliebs, Wolfgang; Girzalsky, Wolfgang; Wilmanns, Matthias; Warscheid, Bettina; Erdmann, Ralf

    2015-10-30

    The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo. PMID:26359497

  14. Human TLR10 is an anti-inflammatory pattern-recognition receptor.

    PubMed

    Oosting, Marije; Cheng, Shih-Chin; Bolscher, Judith M; Vestering-Stenger, Rachel; Plantinga, Theo S; Verschueren, Ineke C; Arts, Peer; Garritsen, Anja; van Eenennaam, Hans; Sturm, Patrick; Kullberg, Bart-Jan; Hoischen, Alexander; Adema, Gosse J; van der Meer, Jos W M; Netea, Mihai G; Joosten, Leo A B

    2014-10-21

    Toll-like receptor (TLR)10 is the only pattern-recognition receptor without known ligand specificity and biological function. We demonstrate that TLR10 is a modulatory receptor with mainly inhibitory effects. Blocking TLR10 by antagonistic antibodies enhanced proinflammatory cytokine production, including IL-1β, specifically after exposure to TLR2 ligands. Blocking TLR10 after stimulation of peripheral blood mononuclear cells with pam3CSK4 (Pam3Cys) led to production of 2,065 ± 106 pg/mL IL-1β (mean ± SEM) in comparison with 1,043 ± 51 pg/mL IL-1β after addition of nonspecific IgG antibodies. Several mechanisms mediate the modulatory effects of TLR10: on the one hand, cotransfection in human cell lines showed that TLR10 acts as an inhibitory receptor when forming heterodimers with TLR2; on the other hand, cross-linking experiments showed specific induction of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra, 16 ± 1.7 ng/mL, mean ± SEM). After cross-linking anti-TLR10 antibody, no production of IL-1β and other proinflammatory cytokines could be found. Furthermore, individuals bearing TLR10 polymorphisms displayed an increased capacity to produce IL-1β, TNF-α, and IL-6 upon ligation of TLR2, in a gene-dose-dependent manner. The modulatory effects of TLR10 are complex, involving at least several mechanisms: there is competition for ligands or for the formation of heterodimer receptors with TLR2, as well as PI3K/Akt-mediated induction of the anti-inflammatory cytokine IL-1Ra. Finally, transgenic mice expressing human TLR10 produced fewer cytokines when challenged with a TLR2 agonist. In conclusion, to our knowledge we demonstrate for the first time that TLR10 is a modulatory pattern-recognition receptor with mainly inhibitory properties.

  15. Dopamine D1 receptor stimulation modulates the formation and retrieval of novel object recognition memory: Role of the prelimbic cortex

    PubMed Central

    Pezze, Marie A.; Marshall, Hayley J.; Fone, Kevin C.F.; Cassaday, Helen J.

    2015-01-01

    Previous studies have shown that dopamine D1 receptor antagonists impair novel object recognition memory but the effects of dopamine D1 receptor stimulation remain to be determined. This study investigated the effects of the selective dopamine D1 receptor agonist SKF81297 on acquisition and retrieval in the novel object recognition task in male Wistar rats. SKF81297 (0.4 and 0.8 mg/kg s.c.) given 15 min before the sampling phase impaired novel object recognition evaluated 10 min or 24 h later. The same treatments also reduced novel object recognition memory tested 24 h after the sampling phase and when given 15 min before the choice session. These data indicate that D1 receptor stimulation modulates both the encoding and retrieval of object recognition memory. Microinfusion of SKF81297 (0.025 or 0.05 μg/side) into the prelimbic sub-region of the medial prefrontal cortex (mPFC) in this case 10 min before the sampling phase also impaired novel object recognition memory, suggesting that the mPFC is one important site mediating the effects of D1 receptor stimulation on visual recognition memory. PMID:26277743

  16. The role of bacteria and pattern-recognition receptors in Crohn's disease.

    PubMed

    Man, Si Ming; Kaakoush, Nadeem O; Mitchell, Hazel M

    2011-03-01

    Crohn's disease is widely regarded as a multifactorial disease, and evidence from human and animal studies suggests that bacteria have an instrumental role in its pathogenesis. Comparison of the intestinal microbiota of patients with Crohn's disease to that of healthy controls has revealed compositional changes. In most studies these changes are characterized by an increase in the abundance of Bacteroidetes and Proteobacteria and a decrease in that of Firmicutes. In addition, a number of specific mucosa-associated bacteria have been postulated to have a role in Crohn's disease, including Mycobacterium avium subspecies paratuberculosis, adherent and invasive Escherichia coli, Campylobacter and Helicobacter species. The association between mutations in pattern-recognition receptors (Toll-like receptors and Nod-like receptors) and autophagy proteins and Crohn's disease provides further evidence to suggest that defective sensing and killing of bacteria may drive the onset of disease. In this Review, we present recent advances in understanding the role of bacteria and the contribution of pattern-recognition receptors and autophagy in the pathogenesis of Crohn's disease.

  17. Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.

    PubMed

    Xu, Rui; de Vries, Robert P; Zhu, Xueyong; Nycholat, Corwin M; McBride, Ryan; Yu, Wenli; Paulson, James C; Wilson, Ian A

    2013-12-01

    The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu(226), a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.

  18. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  19. Expanding the universe of cytokines and pattern recognition receptors: galectins and glycans in innate immunity.

    PubMed

    Cerliani, Juan P; Stowell, Sean R; Mascanfroni, Iván D; Arthur, Connie M; Cummings, Richard D; Rabinovich, Gabriel A

    2011-02-01

    Effective immunity relies on the recognition of pathogens and tumors by innate immune cells through diverse pattern recognition receptors (PRRs) that lead to initiation of signaling processes and secretion of pro- and anti-inflammatory cytokines. Galectins, a family of endogenous lectins widely expressed in infected and neoplastic tissues have emerged as part of the portfolio of soluble mediators and pattern recognition receptors responsible for eliciting and controlling innate immunity. These highly conserved glycan-binding proteins can control immune cell processes through binding to specific glycan structures on pathogens and tumors or by acting intracellularly via modulation of selective signaling pathways. Recent findings demonstrate that various galectin family members influence the fate and physiology of different innate immune cells including polymorphonuclear neutrophils, mast cells, macrophages, and dendritic cells. Moreover, several pathogens may actually utilize galectins as a mechanism of host invasion. In this review, we aim to highlight and integrate recent discoveries that have led to our current understanding of the role of galectins in host-pathogen interactions and innate immunity. Challenges for the future will embrace the rational manipulation of galectin-glycan interactions to instruct and shape innate immunity during microbial infections, inflammation, and cancer.

  20. A Temporally Distinct Role for Group I and Group II Metabotropic Glutamate Receptors in Object Recognition Memory

    ERIC Educational Resources Information Center

    Brown, Malcolm Watson; Warburton, Elizabeth Clea; Barker, Gareth Robert Isaac; Bashir, Zafar Iqbal

    2006-01-01

    Recognition memory, involving the ability to discriminate between a novel and familiar object, depends on the integrity of the perirhinal cortex (PRH). Glutamate, the main excitatory neurotransmitter in the cortex, is essential for many types of memory processes. Of the subtypes of glutamate receptor, metabotropic receptors (mGluRs) have received…

  1. Variation in the Oxytocin Receptor Gene Is Associated with Face Recognition and its Neural Correlates

    PubMed Central

    Westberg, Lars; Henningsson, Susanne; Zettergren, Anna; Svärd, Joakim; Hovey, Daniel; Lin, Tian; Ebner, Natalie C.; Fischer, Håkan

    2016-01-01

    The ability to recognize faces is crucial for daily social interactions. Recent studies suggest that intranasal oxytocin administration improves social recognition in humans. Oxytocin signaling in the amygdala plays an essential role for social recognition in mice, and oxytocin administration has been shown to influence amygdala activity in humans. It is therefore possible that the effects of oxytocin on human social recognition depend on mechanisms that take place in the amygdala—a central region for memory processing also in humans. Variation in the gene encoding the oxytocin receptor (OXTR) has been associated with several aspects of social behavior. The present study examined the potential associations between nine OXTR polymorphisms, distributed across the gene, and the ability to recognize faces, as well as face-elicited amygdala activity measured by functional magnetic resonance imaging (fMRI) during incidental encoding of faces. The OXTR 3′ polymorphism rs7632287, previously related to social bonding behavior and autism risk, was associated with participants’ ability to recognize faces. Carriers of the GA genotype, associated with enhanced memory, displayed higher amygdala activity during face encoding compared to carriers of the GG genotype. In line with work in rodents, these findings suggest that, in humans, naturally occurring endogenous modulation of OXTR function affects social recognition through an amygdala-dependent mechanism. These findings contribute to the understanding of how oxytocin regulates human social behaviors. PMID:27713694

  2. Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

    PubMed Central

    Maqbool, A; Saitoh, H; Franceschetti, M; Stevenson, CEM; Uemura, A; Kanzaki, H; Kamoun, S; Terauchi, R; Banfield, MJ

    2015-01-01

    Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001 PMID:26304198

  3. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

    PubMed Central

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-01-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c+, CD141+ and CD16+ myeloid DCs and CD123+ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141+ DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  4. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

    PubMed

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-06-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  5. Metal Oxide Nanosensors Using Polymeric Membranes, Enzymes and Antibody Receptors as Ion and Molecular Recognition Elements

    PubMed Central

    Willander, Magnus; Khun, Kimleang; Ibupoto, Zafar Hussain

    2014-01-01

    The concept of recognition and biofunctionality has attracted increasing interest in the fields of chemistry and material sciences. Advances in the field of nanotechnology for the synthesis of desired metal oxide nanostructures have provided a solid platform for the integration of nanoelectronic devices. These nanoelectronics-based devices have the ability to recognize molecular species of living organisms, and they have created the possibility for advanced chemical sensing functionalities with low limits of detection in the nanomolar range. In this review, various metal oxides, such as ZnO-, CuO-, and NiO-based nanosensors, are described using different methods (receptors) of functionalization for molecular and ion recognition. These functionalized metal oxide surfaces with a specific receptor involve either a complex formation between the receptor and the analyte or an electrostatic interaction during the chemical sensing of analytes. Metal oxide nanostructures are considered revolutionary nanomaterials that have a specific surface for the immobilization of biomolecules with much needed orientation, good conformation and enhanced biological activity which further improve the sensing properties of nanosensors. Metal oxide nanostructures are associated with certain unique optical, electrical and molecular characteristics in addition to unique functionalities and surface charge features which shows attractive platforms for interfacing biorecognition elements with effective transducing properties for signal amplification. There is a great opportunity in the near future for metal oxide nanostructure-based miniaturization and the development of engineering sensor devices. PMID:24841244

  6. T cell recognition of weak ligands: roles of signaling, receptor number, and affinity

    PubMed Central

    Edwards, Lindsay J.

    2011-01-01

    T cell recognition of antigen is a crucial aspect of the adaptive immune response. One of the most common means of pathogen immune evasion is mutation of T cell epitopes. T cell recognition of such ligands can result in a variety of outcomes including activation, apoptosis and anergy. The ability of a given T cell to respond to a specific peptide–MHC ligand is regulated by a number of factors, including the affinity, on- and off-rates and half-life of the TCR–peptide–MHC interaction. Interaction of T cells with low-potency ligands results in unique signaling patterns and requires engagement with a larger number of T cell receptors than agonist ligands. This review will address these aspects of T cell interaction with weak ligands and the ways in which these ligands have been utilized therapeutically. PMID:21365321

  7. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    SciTech Connect

    Pioszak, Augen A.; Xu, H. Eric

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  8. Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor

    SciTech Connect

    Whitelaw, M.; Pongratz, I.; Wilhelmsson, A.; Gustafsson, J.; Poellinger, L. )

    1993-04-01

    Signal transduction by dioxins is mediated by the intracellular dioxin or aryl hydrocarbon receptor. This receptor binds dioxin and its planar aromatic congeners in a saturable manner with high affinity. The extreme toxicity of dioxin has been demonstrated in animals but not in humans. In animals, dioxin causes thymic wasting, immune suppression, severe epithelial disorders and tumor promotion. On a molecular level, dioxins are inducers of transcription of a battery of target genes encoding xenobiotic metabolizing enzymes. Dioxin also appears to transcriptionally regulate the expression of the growth modulatory genes for interleukin-1 Beta and plasminogen activator inhibitor-2. The dioxin induction response is mediated by single or multiple copies of dioxin-inducible transcriptional control elements in target promoters. The research data detailed in this paper examines the ligand-dependent recruitment of the Arnt coregulator which determines DNA recognition by the dioxin receptor. This data suggests that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt and that the dioxin receptor system provides the first example of signal-controlled dimerization of bHLH factors.

  9. Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline

    PubMed Central

    Trdá, Lucie; Boutrot, Freddy; Claverie, Justine; Brulé, Daphnée; Dorey, Stephan; Poinssot, Benoit

    2015-01-01

    Plants are continuously monitoring the presence of microorganisms to establish an adapted response. Plants commonly use pattern recognition receptors (PRRs) to perceive microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) which are microorganism molecular signatures. Located at the plant plasma membrane, the PRRs are generally receptor-like kinases (RLKs) or receptor-like proteins (RLPs). MAMP detection will lead to the establishment of a plant defense program called MAMP-triggered immunity (MTI). In this review, we overview the RLKs and RLPs that assure early recognition and control of pathogenic or beneficial bacteria. We also highlight the crucial function of PRRs during plant-microbe interactions, with a special emphasis on the receptors of the bacterial flagellin and peptidoglycan. In addition, we discuss the multiple strategies used by bacteria to evade PRR-mediated recognition. PMID:25904927

  10. Molecular Recognition Analyzed by Docking Simulations: The Aspartate Receptor and Isocitrate Dehydrogenase from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Stoddard, Barry L.; Koshland, Daniel E., Jr.

    1993-02-01

    Protein docking protocols are used for the prediction of both small molecule binding to DNA and protein macromolecules and of complexes between macromolecules. These protocols are becoming increasingly automated and powerful tools for computer-aided drug design. We review the basic methodologies and strategies used for analyzing molecular recognition by computer docking algorithms and discuss recent experiments in which (i) substrate and substrate analogues are docked to the active site of isocitrate dehydrogenase and (ii) maltose binding protein is docked to the extracellular domain of the receptor, which signals maltose chemotaxis.

  11. The role of the IAP E3 ubiquitin ligases in regulating pattern-recognition receptor signalling.

    PubMed

    Vandenabeele, Peter; Bertrand, Mathieu J M

    2012-12-01

    An inflammatory response is initiated when innate immune pattern-recognition receptors (PRRs) expressed by different cell types detect constituents of invading microorganisms and endogenous intracellular molecules released by dying cells. The intracellular cascades activated by PRRs induce the expression and maturation of inflammatory molecules that coordinate the removal of the infectious agents and of the infected or damaged cells. In this Review, we discuss the findings implicating members of the inhibitor of apoptosis protein (IAP) family in the ubiquitylation-dependent regulation of PRR signalling. Understanding the role of IAPs in innate immunity may open new therapeutic perspectives for the treatment of PRR-dependent inflammatory diseases.

  12. Immunopathological roles of cytokines, chemokines, signaling molecules, and pattern-recognition receptors in systemic lupus erythematosus.

    PubMed

    Yu, Shui-Lian; Kuan, Woon-Pang; Wong, Chun-Kwok; Li, Edmund K; Tam, Lai-Shan

    2012-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease with unknown etiology affecting more than one million individuals each year. It is characterized by B- and T-cell hyperactivity and by defects in the clearance of apoptotic cells and immune complexes. Understanding the complex process involved and the interaction between various cytokines, chemokines, signaling molecules, and pattern-recognition receptors (PRRs) in the immune pathways will provide valuable information on the development of novel therapeutic targets for treating SLE. In this paper, we review the immunopathological roles of novel cytokines, chemokines, signaling molecules, PRRs, and their interactions in immunoregulatory networks and suggest how their disturbances may implicate pathological conditions in SLE.

  13. Oxytocin and the oxytocin receptor underlie intrastrain, but not interstrain, social recognition.

    PubMed

    Macbeth, A H; Lee, H-J; Edds, J; Young, W S

    2009-07-01

    We studied three lines of oxytocin (Oxt) and oxytocin receptor (Oxtr) knockout (KO) male mice [Oxt(-/-), total Oxtr(-/-) and partial forebrain Oxtr (Oxtr(FB/FB))] with established deficits in social recognition to further refine our understanding of their deficits with regard to stimulus female's strain. We used a modified social discrimination paradigm in which subjects are singly housed only for the duration of the test. Additionally, stimulus females are singly housed throughout testing and are presented within corrals for rapid comparison of investigation by subject males. Wild-type (WT) males from all three lines discriminated between familiar and novel females of three different strains (C57BL/6, BALB/c and Swiss-Webster). No KO males discriminated between familiar and novel BALB/c or C57BL/6 females. Male Oxt(-/-) and Oxtr(-/-) mice, but not Oxtr(FB/FB) mice, discriminated between familiar and novel Swiss-Webster females. As this might indicate a global deficit in individual recognition for Oxtr(FB/FB) males, we examined their ability to discriminate between females from different strains and compared performance with Oxtr(-/-) males. WT and KO males from both lines were able to distinguish between familiar and novel females from different strains, indicating the social recognition deficit is not universal. Instead, we hypothesize that the Oxtr is involved in 'fine' intrastrain recognition, but is less important in 'broad' interstrain recognition. We also present the novel finding of decreased investigation across tests, which is likely an artifact of repeated testing and not because of stimulus female's strain or age of subject males.

  14. The response of human dendritic cells to co-ligation of pattern-recognition receptors.

    PubMed

    Dzopalic, Tanja; Rajkovic, Ivan; Dragicevic, Ana; Colic, Miodrag

    2012-04-01

    Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.

  15. Ameliorative effect of membrane-associated estrogen receptor G protein coupled receptor 30 activation on object recognition memory in mouse models of Alzheimer's disease.

    PubMed

    Kubota, Takashi; Matsumoto, Hiroshi; Kirino, Yutaka

    2016-07-01

    Membrane-associated estrogen receptor "G protein-coupled receptor 30" (GPR30) has been implicated in spatial recognition memory and protection against neuronal death. The present study investigated the role of GPR30 in object recognition memory in an Alzheimer's disease (AD) mouse model (5XFAD) by using novel object recognition (NOR) test. Impairment of long-term (24 h) recognition memory was observed in both male and female 5XFAD mice. Selective GPR30 agonist, G-1, ameliorated this impairment in female 5XFAD mice, but not in male mice. Our study demonstrated the ameliorative role of GPR30 in NOR memory impaired by AD pathology in female mice. PMID:27423484

  16. Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition.

    PubMed

    Voogdt, Carlos G P; Bouwman, Lieneke I; Kik, Marja J L; Wagenaar, Jaap A; van Putten, Jos P M

    2016-01-01

    Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions.

  17. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    SciTech Connect

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  18. Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition

    PubMed Central

    Voogdt, Carlos G. P.; Bouwman, Lieneke I.; Kik, Marja J. L.; Wagenaar, Jaap A.; van Putten, Jos P. M.

    2016-01-01

    Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions. PMID:26738735

  19. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors.

    PubMed

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F

    2016-01-26

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å(2) of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition. PMID:26744311

  20. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors.

    PubMed

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F

    2016-01-26

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å(2) of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition.

  1. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors

    PubMed Central

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F.

    2016-01-01

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å2 of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition. PMID:26744311

  2. Molecular Recognition by a Polymorphic Cell Surface Receptor Governs Cooperative Behaviors in Bacteria

    PubMed Central

    Dey, Arup; Wall, Daniel

    2013-01-01

    Cell-cell recognition is a fundamental process that allows cells to coordinate multicellular behaviors. Some microbes, such as myxobacteria, build multicellular fruiting bodies from free-living cells. However, how bacterial cells recognize each other by contact is poorly understood. Here we show that myxobacteria engage in recognition through interactions between TraA cell surface receptors, which leads to the fusion and exchange of outer membrane (OM) components. OM exchange is shown to be selective among 17 environmental isolates, as exchange partners parsed into five major recognition groups. TraA is the determinant of molecular specificity because: (i) exchange partners correlated with sequence conservation within its polymorphic PA14-like domain and (ii) traA allele replacements predictably changed partner specificity. Swapping traA alleles also reprogrammed social interactions among strains, including the regulation of motility and conferred immunity from inter-strain killing. We suggest that TraA helps guide the transition of single cells into a coherent bacterial community, by a proposed mechanism that is analogous to mitochondrial fusion and fission cycling that mixes contents to establish a homogenous population. In evolutionary terms, traA functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment. PMID:24244178

  3. Different roles for M1 and M2 receptors within perirhinal cortex in object recognition and discrimination.

    PubMed

    Bartko, Susan J; Winters, Boyer D; Saksida, Lisa M; Bussey, Timothy J

    2014-04-01

    Recognition and discrimination of objects and individuals are critical cognitive faculties in both humans and non-human animals, and cholinergic transmission has been shown to be essential for both of these functions. In the present study we focused on the role of M1 and M2 muscarinic receptors in perirhinal cortex (PRh)-dependent object recognition and discrimination. The selective M1 antagonists pirenzepine and the snake toxin MT-7, and a selective M2 antagonist, AF-DX 116, were infused directly into PRh. Pre-sample infusions of both pirenzepine and AF-DX 116 significantly impaired object recognition memory in a delay-dependent manner. However, pirenzepine and MT-7, but not AF-DX 116, impaired oddity discrimination performance in a perceptual difficulty-dependent manner. The findings indicate distinct functions for M1 and M2 receptors in object recognition and discrimination.

  4. Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris.

    PubMed

    Zhang, Huan; Kong, Pengfei; Wang, Lingling; Zhou, Zhi; Yang, Jialong; Zhang, Ying; Qiu, Limei; Song, Linsheng

    2010-07-01

    C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity. The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues. The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively. But its expression level did not change significantly during peptidoglycan (PGN) stimulation. The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3). The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way. The agglutinating activity could be inhibited by d-mannose, LPS and glucan, but not by d-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs. PMID:20211738

  5. Macrophage recognition of toxic advanced glycosylation end products through the macrophage surface-receptor nucleolin.

    PubMed

    Miki, Yuichi; Dambara, Hikaru; Tachibana, Yoshihiro; Hirano, Kazuya; Konishi, Mio; Beppu, Masatoshi

    2014-01-01

    Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.

  6. Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition

    SciTech Connect

    Verdino, P.; Aldag, C.; Hilvert, D.; Wilson, I.A.

    2009-05-26

    Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu{sup H47}Trp and Arg{sup H100}Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu{sup H47}Trp/Arg{sup H100}Trp double mutant (1E9dm) as an unliganded Fab at 2.05 {angstrom} resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 {angstrom}. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.

  7. Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones.

    PubMed

    Tóth, Gergő; Baska, Ferenc; Schretner, András; Rácz, Akos; Noszál, Béla

    2013-09-01

    Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications. PMID:23907234

  8. Selective recognition of neutral guests in an aqueous medium by a biomimetic calix[6]cryptamide receptor.

    PubMed

    Lascaux, Angélique; De Leener, Gaël; Fusaro, Luca; Topić, Filip; Rissanen, Kari; Luhmer, Michel; Jabin, Ivan

    2016-01-14

    The design of artificial receptors that can efficiently work in water is a challenging research area. A possible biomimetic approach for the elaboration of such receptors consists of associating a hydrophobic cavity with a polar polyfunctional binding site. On this basis, a hydrophilic calix[6]cryptamide decorated with oligo(ethylene glycol) units (i.e. 8) was synthesized through an efficient [1 + 1] macrocyclization reaction as the key-step. The complexation of neutral molecules was evaluated by NMR spectroscopy through competition experiments either in apolar or aqueous media. In both media, host 8 can bind neutral species that display H-bonding acceptor and donor groups such as amides or ureas. Interestingly, the most polar and acidic molecule is the best guest in chloroform and the worst one in an aqueous medium, highlighting the importance of the environment. As shown by NMR and X-ray diffraction data, the mode of recognition involves a complementary DAAAD-ADDDA quintuple H-bonding array between the binding partners as well as multiple CH-π interactions. A comparison of this calix[6]arene-based host-guest system with the binding site of biotin-binding proteins shows strong similarities. Besides, the acid-base control of the binding properties of receptor 8 in aqueous media is highly reminiscent of allosteric processes encountered in natural systems. PMID:26580493

  9. Enantioselective recognition of mono-demethylated methoxychlor metabolites by the estrogen receptor.

    PubMed

    Miyashita, Masahiro; Shimada, Takahiro; Nakagami, Shizuka; Kurihara, Norio; Miyagawa, Hisashi; Akamatsu, Miki

    2004-02-01

    Metabolites of methoxychlor such as 2-(p-hydroxyphenyl)-2-(p-methoxyphenyl)-1,1,1-trichloroethane (mono-OH-MXC) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (bis-OH-MXC), have estrogenic activity. Mono-OH-MXC is a chiral compound in which the carbon atom bridging two benzene rings is the chiral centre. In previous studies the estrogenic activity of racemic mono-OH-MXC has been measured, and the activity of each enantiomer of this compound has not yet been elucidated. In this study, we evaluated the estrogen receptor-binding activity of each enantiomer of mono-OH-MXC to clarify the enantioselective recognition by the estrogen receptor. (S)-mono-OH-MXC showed 3-fold higher binding activity than that of the (R) enantiomer. The activity of bis-OH-MXC was only 1.7-fold higher than that of (S)-mono-OH-MXC. This result suggests that the one hydroxy group and the orientation of the CCl3 group of mono- and bis-OH-MXCs are important for the interaction with the estrogen receptor. The result also points out the estrogenic activity of methoxychlor after metabolic activation in vivo, which predominantly produces the (S)-mono-OH-MXC, may be higher than estimated from the in vitro activity of racemic mixtures.

  10. Recognition and sequestration of ω-fatty acids by a cavitand receptor

    PubMed Central

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-01-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring “prestacked” aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules—unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide—from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules. PMID:26305974

  11. Recognition and sequestration of ω-fatty acids by a cavitand receptor.

    PubMed

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-09-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring "prestacked" aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules-unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide-from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules.

  12. Selective recognition of neutral guests in an aqueous medium by a biomimetic calix[6]cryptamide receptor.

    PubMed

    Lascaux, Angélique; De Leener, Gaël; Fusaro, Luca; Topić, Filip; Rissanen, Kari; Luhmer, Michel; Jabin, Ivan

    2016-01-14

    The design of artificial receptors that can efficiently work in water is a challenging research area. A possible biomimetic approach for the elaboration of such receptors consists of associating a hydrophobic cavity with a polar polyfunctional binding site. On this basis, a hydrophilic calix[6]cryptamide decorated with oligo(ethylene glycol) units (i.e. 8) was synthesized through an efficient [1 + 1] macrocyclization reaction as the key-step. The complexation of neutral molecules was evaluated by NMR spectroscopy through competition experiments either in apolar or aqueous media. In both media, host 8 can bind neutral species that display H-bonding acceptor and donor groups such as amides or ureas. Interestingly, the most polar and acidic molecule is the best guest in chloroform and the worst one in an aqueous medium, highlighting the importance of the environment. As shown by NMR and X-ray diffraction data, the mode of recognition involves a complementary DAAAD-ADDDA quintuple H-bonding array between the binding partners as well as multiple CH-π interactions. A comparison of this calix[6]arene-based host-guest system with the binding site of biotin-binding proteins shows strong similarities. Besides, the acid-base control of the binding properties of receptor 8 in aqueous media is highly reminiscent of allosteric processes encountered in natural systems.

  13. Poly I:C-induced tumor cell apoptosis mediated by pattern-recognition receptors.

    PubMed

    Zhao, Xiangzhong; Ai, Miao; Guo, Yuqi; Zhou, Xianbin; Wang, Li; Li, Xia; Yao, Chengfang

    2012-11-01

    Poly I:C is a synthetic dsRNA that can imitate a viral infection and elicit host immune responses by triggering specific pattern-recognition receptors (PRRs) such as toll-like receptor 3 and retinoic acid inducible gene I(RIG-I)-like receptors, including RIG-I and melanoma differentiation-associated gene 5. Activation of these PRRs by poly I:C triggers a signal transduction cascade that results in the activation of NF-κB and production of type I interferon. Poly I:C has been used as a vaccine adjuvant for cancer immunotherapy for several decades. Evidence from recent studies indicates that poly I:C can directly induce apoptosis in several types of tumor cells, thus providing a new therapeutic approach for cancer treatment. However, the molecular mechanism underlying the induction of apoptosis by poly I:C is still unclear. In this review, we summarize the current knowledge of poly I:C-induced tumor cell apoptosis, focusing on the key molecules and pathways involved in this process.

  14. Kappa Opioid Receptor-Mediated Disruption of Novel Object Recognition: Relevance for Psychostimulant Treatment

    PubMed Central

    Paris, Jason J.; Reilley, Kate J.; McLaughlin, Jay P.

    2012-01-01

    Kappa opioid receptor (KOR) agonists are potentially valuable as therapeutics for the treatment of psychostimulant reward as they suppress dopamine signaling in reward circuitry to repress drug seeking behavior. However, KOR agonists are also associated with sedation and cognitive dysfunction. The extent to which learning and memory disruption or hypolocomotion underlie KOR agonists’ role in counteracting the rewarding effects of psychostimulants is of interest. C57BL/6J mice were pretreated with vehicle (saline, 0.9%), the KOR agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), or the peripherally-restricted agonist D-Phe-D-Phe-D-lle-D-Arg- NH2 (ffir-NH2), through central (i.c.v.) or peripheral (i.p.) routes of administration. Locomotor activity was assessed via activity monitoring chambers and rotorod. Cognitive performance was assessed in a novel object recognition task. Prolonged hypolocomotion was observed following administration of 1.0 and 10.0, but not 0.3 mg/kg U50,488. Central, but not peripheral, administration of ffir-NH2 (a KOR agonist that does not cross the blood-brain barrier) also reduced motor behavior. Systemic pretreatment with the low dose of U50,488 (0.3 mg/kg, i.p.) significantly impaired performance in the novel object recognition task. Likewise, ffir-NH2 significantly reduced novel object recognition after central (i.c.v.), but not peripheral (i.p.), administration. U50,488- and ffir-NH2-mediated deficits in novel object recognition were prevented by pretreatment with KOR antagonists. Cocaine-induced conditioned place preference was subsequently assessed and was reduced by pretreatment with U50,488 (0.3 mg/kg, i.p.). Together, these results suggest that the activation of centrally-located kappa opioid receptors may induce cognitive and mnemonic disruption independent of hypolocomotor effects which may contribute to the KOR-mediated suppression of psychostimulant reward. PMID:22900234

  15. Signal recognition particle receptor is a complex that contains two distinct polypeptide chains

    SciTech Connect

    Tajima, S.; Lauffer, L.; Rath, V.L.; Walter, P.

    1986-10-01

    Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here the authors report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (..cap alpha..-subunit, SR..cap alpha..), a 30-kD ..beta..-subunit SR..beta..). When SRP receptor was purified by SRP-Sepharose affinity chromatography, they observed the co-purification of two other ER membrane proteins. Both proteins are approx.30 kD in size and are immunologically distinct from each other, as well as from SR..cap alpha.. and SRP proteins. One of the 30-kD proteins (SR..beta..) forms a tight complex with SR..cap alpha.. in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR..cap alpha.. or SR..beta... Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. They therefore conclude that SR..beta.. is an integral component of SRP receptor. The presence of SR..beta.. was previously masked by proteolytic breakdown products of SR..cap alpha.. observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR..cap alpha... Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR..cap alpha.. and SR..beta... The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

  16. Recognition of lipid A variants by the TLR4-MD-2 receptor complex

    PubMed Central

    Maeshima, Nina; Fernandez, Rachel C.

    2012-01-01

    Lipopolysaccharide (LPS) is a component of the outer membrane of almost all Gram-negative bacteria and consists of lipid A, core sugars, and O-antigen. LPS is recognized by Toll-like receptor 4 (TLR4) and MD-2 on host innate immune cells and can signal to activate the transcription factor NFκB, leading to the production of pro-inflammatory cytokines that initiate and shape the adaptive immune response. Most of what is known about how LPS is recognized by the TLR4-MD-2 receptor complex on animal cells has been studied using Escherichia coli lipid A, which is a strong agonist of TLR4 signaling. Recent work from several groups, including our own, has shown that several important pathogenic bacteria can modify their LPS or lipid A molecules in ways that significantly alter TLR4 signaling to NFκB. Thus, it has been hypothesized that expression of lipid A variants is one mechanism by which pathogens modulate or evade the host immune response. Additionally, several key differences in the amino acid sequences of human and mouse TLR4-MD-2 receptors have been shown to alter the ability to recognize these variations in lipid A, suggesting a host-specific effect on the immune response to these pathogens. In this review, we provide an overview of lipid A variants from several human pathogens, how the basic structure of lipid A is recognized by mouse and human TLR4-MD-2 receptor complexes, as well as how alteration of this pattern affects its recognition by TLR4 and impacts the downstream immune response. PMID:23408095

  17. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  18. The selectivity of water-based pyrophosphate recognition is tuned by metal substitution in dimetallic receptors.

    PubMed

    Svane, Simon; Kjeldsen, Frank; McKee, Vickie; McKenzie, Christine J

    2015-07-14

    The three dimetallic compounds [Ga2(bpbp)(OH)2(H2O)2](ClO4)3, [In2(bpbp)(CH3CO2)2](ClO4)3 and [Zn2(bpbp)(HCO2)2](ClO4) (bpbp(-) = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate) were evaluated as stable solid state precursors for reactive solution state receptors to use for the recognition of the biologically important anion pyrophosphate in water at neutral pH. Indicator displacement assays using in situ generated complex-pyrocatechol violet adducts, {M2(bpbp)(HxPV)}(n+) M = Ga(3+), In(3+), Zn(2+), were tested for selectivity in their reactions with a series of common anions: pyrophosphate, phosphate, ATP, arsenate, nitrate, perchlorate, chloride, sulfate, formate, carbonate and acetate. The receptor employing Ga(3+) showed a slow but visually detectable response (blue to yellow) in the presence of one equivalent of pyrophosphate but no response to any other anion, even when they were present in much higher concentrations. The systems based on In(3+) or Zn(2+) show less selectivity in accord with visibly discernible responses to several of the anions. These results demonstrate a facile method for increasing anion selectivity without modification of an organic dinucleating ligand scaffold. The comfortable supramolecular recognition of pyrophosphate by the dimetallic complexes is demonstrated by the single crystal X-ray structure of [Ga2(bpbp)(HP2O7)](ClO4)2 in which the pyrophosphate is coordinated to the two gallium ions via four of its oxygen atoms.

  19. The selectivity of water-based pyrophosphate recognition is tuned by metal substitution in dimetallic receptors.

    PubMed

    Svane, Simon; Kjeldsen, Frank; McKee, Vickie; McKenzie, Christine J

    2015-07-14

    The three dimetallic compounds [Ga2(bpbp)(OH)2(H2O)2](ClO4)3, [In2(bpbp)(CH3CO2)2](ClO4)3 and [Zn2(bpbp)(HCO2)2](ClO4) (bpbp(-) = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate) were evaluated as stable solid state precursors for reactive solution state receptors to use for the recognition of the biologically important anion pyrophosphate in water at neutral pH. Indicator displacement assays using in situ generated complex-pyrocatechol violet adducts, {M2(bpbp)(HxPV)}(n+) M = Ga(3+), In(3+), Zn(2+), were tested for selectivity in their reactions with a series of common anions: pyrophosphate, phosphate, ATP, arsenate, nitrate, perchlorate, chloride, sulfate, formate, carbonate and acetate. The receptor employing Ga(3+) showed a slow but visually detectable response (blue to yellow) in the presence of one equivalent of pyrophosphate but no response to any other anion, even when they were present in much higher concentrations. The systems based on In(3+) or Zn(2+) show less selectivity in accord with visibly discernible responses to several of the anions. These results demonstrate a facile method for increasing anion selectivity without modification of an organic dinucleating ligand scaffold. The comfortable supramolecular recognition of pyrophosphate by the dimetallic complexes is demonstrated by the single crystal X-ray structure of [Ga2(bpbp)(HP2O7)](ClO4)2 in which the pyrophosphate is coordinated to the two gallium ions via four of its oxygen atoms. PMID:26057368

  20. Neural Androgen Receptors Modulate Gene Expression and Social Recognition But Not Social Investigation

    PubMed Central

    Karlsson, Sara A.; Studer, Erik; Kettunen, Petronella; Westberg, Lars

    2016-01-01

    The role of sex and androgen receptors (ARs) for social preference and social memory is rather unknown. In this study of mice we compared males, females and males lacking ARs specifically in the nervous system, ARNesDel, with respect to social preference, assessed with the three-chambered apparatus test, and social recognition, assessed with the social discrimination procedure. In the social discrimination test we also evaluated the tentative importance of the sex of the stimulus animal. Novel object recognition and olfaction were investigated to complement the results from the social tests. Gene expression analysis was performed to reveal molecules involved in the effects of sex and androgens on social behaviors. All three test groups showed social preference in the three-chambered apparatus test. In both social tests an AR-independent sexual dimorphism was seen in the persistence of social investigation of female conspecifics, whereas the social interest toward male stimuli mice was similar in all groups. Male and female controls recognized conspecifics independent of their sex, whereas ARNesDel males recognized female but not male stimuli mice. Moreover, the non-social behaviors were not affected by AR deficiency. The gene expression analyses of hypothalamus and amygdala indicated that Oxtr, Cd38, Esr1, Cyp19a1, Ucn3, Crh, and Gtf2i were differentially expressed between the three groups. In conclusion, our results suggest that ARs are required for recognition of male but not female conspecifics, while being dispensable for social investigation toward both sexes. In addition, the AR seems to regulate genes related to oxytocin, estrogen and William’s syndrome. PMID:27014003

  1. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  2. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors

    PubMed Central

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H. P.; Mathew, Paul; Navone, Nora M.; Lin, Sue-Hwa; Logothetis, Christopher J.; Rietz, Anna Cecilia; Dobroff, Andrey S.; Proneth, Bettina; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand–receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  3. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors.

    PubMed

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H P; Mathew, Paul; Navone, Nora M; Lin, Sue-Hwa; Logothetis, Christopher J; Rietz, Anna Cecilia; Dobroff, Andrey S; Proneth, Bettina; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih

    2015-03-24

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  4. Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

    PubMed Central

    Lee, Benhur; Pernet, Olivier; Ahmed, Asim A.; Zeltina, Antra; Beaty, Shannon M.; Bowden, Thomas A.

    2015-01-01

    The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations. PMID:25825759

  5. Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

    NASA Astrophysics Data System (ADS)

    Li, Zhen; Deng, Si-Si; Zang, Yi; Gu, Zhen; He, Xiao-Peng; Chen, Guo-Rong; Chen, Kaixian; James, Tony D.; Li, Jia; Long, Yi-Tao

    2013-07-01

    Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

  6. Chiral diaminopyrrolic receptors for selective recognition of mannosides, part 2: a 3D view of the recognition modes by X-ray, NMR spectroscopy, and molecular modeling.

    PubMed

    Ardá, Ana; Cañada, F Javier; Nativi, Cristina; Francesconi, Oscar; Gabrielli, Gabriele; Ienco, Andrea; Jiménez-Barbero, Jesús; Roelens, Stefano

    2011-04-18

    The structural features of a representative set of five complexes of octyl α- and β-mannosides with some members of a new generation of chiral tripodal diaminopyrrolic receptors, namely, (R)-5 and (S)- and (R)-7, have been investigated in solution and in the solid state by a combined X-ray, NMR spectroscopy, and molecular modeling approach. In the solid state, the binding arms of the free receptors 7 delimit a cleft in which two solvent molecules are hydrogen bonded to the pyrrolic groups and to the benzenic scaffold. In a polar solvent (CD(3)CN), chemical shift and intermolecular NOE data, assisted by molecular modeling calculations, ascertained the binding modes of the interaction between the receptor and the glycoside for these complexes. Although a single binding mode was found to adequately describe the complex of the acyclic receptor 5 with the α-mannoside, for the complexes of the cyclic receptors 7 two different binding modes were required to simultaneously fit all the experimental data. In all cases, extensive binding through hydrogen bonding and CH-π interactions is responsible for the affinities measured in the same solvent. Furthermore, the binding modes closely account for the recognition preferences observed toward the anomeric glycosides and for the peculiar enantiodiscrimination properties exhibited by the chiral receptors.

  7. Ion pair recognition receptor based on an unsymmetrically 1,1'-disubstituted ferrocene-triazole derivative.

    PubMed

    Otón, Francisco; González, María del Carmen; Espinosa, Arturo; Ramírez de Arellano, Carmen; Tárraga, Alberto; Molina, Pedro

    2012-11-16

    The ferrocene-triazole derivative 4, available from 1,1'-bis(diazido)ferrocene by sequential functionalization through click-type chemistry and the Staudinger reaction, is elaborated as a lab-on-a-molecule for the selective sensing of HP(2)O(7)(3-) and Hg(2+). Receptor 4 behaves as a ratiometric fluorescent probe for HP(2)O(7)(3-) with a good selectivity over other anions, whereas in the presence of Hg(2+) it modifies the fluorescent emission of the pyrene unit, acting as a selective on-off fluorescent sensor for Hg(2+) with a low detection limit. The most salient feature of compound 4 is its behavior as an excellent electrooptical ion pair recognition receptor able to simultaneously recognize Pb(2+) cations in the presence of HP(2)O(7)(3-) anion through multichannel perturbations of the redox potential of the ferrocene unit, the emission spectrum, and a noticeable color change from yellow to green.

  8. Deramciclane improves object recognition in rats: potential role of NMDA receptors.

    PubMed

    Kertész, Szabolcs; Kapus, Gábor; Gacsályi, István; Lévay, György

    2010-02-01

    The cognition-enhancing properties of deramciclane (N,N-dimethyl-2-([(1R,4R,6S)-1,7,7-trimethyl-6-phenyl-6-bicyclo[2.2.1]heptanyl]oxy)ethanamine) and memantine (3,5-dimethyl-tricyclo[3.3.1.1(3,7)]decylamine-3,5-dimethyladamantan-1-amine) were evaluated in the novel object recognition (OR) test in the rat, while their effect in comparison with other N-methyl-D-aspartate (NMDA) receptor blockers such us MK-801 ([+]-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate) and CPP ([+/-]-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) on NMDA-evoked spreading depression (SD) was investigated in the chicken retina, in vitro. In the OR test, pretreatment of rats with either deramciclane (30 mg/kg p.o.) or memantine (10 and 30 mg/kg, p.o.) resulted in preference for the novel object, compared to the familiar one, indicating procognitive activity of the compounds. In the in vitro studies memantine (10-30 M), or deramciclane (30-100 M) as well as CPP (0.1-1 M), MK-801 (0.3-1 M), concentration-dependently inhibited NMDA evoked SD. Furthermore, the inhibitory effect of memantine, deramciclane and MK-801 was activity-dependent. These results support the role of NMDA receptors in the procognitive effect of deramciclane.

  9. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    PubMed

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  10. Anion recognition by simple chromogenic and chromo-fluorogenic salicylidene Schiff base or reduced-Schiff base receptors.

    PubMed

    Dalapati, Sasanka; Jana, Sankar; Guchhait, Nikhil

    2014-08-14

    This review contains extensive application of anion sensing ability of salicylidene type Schiff bases and their reduced forms having various substituents with respect to phenolic OH group. Some of these molecular systems behave as receptor for recognition or sensing of various anions in organic or aqueous-organic binary solvent mixture as well as in the solid supported test kits. Development of Schiff base or reduced Schiff base receptors for anion recognition event is commonly based on the theory of hydrogen bonding interaction or deprotonation of phenolic -OH group. The process of charge transfer (CT) or inhibition of excited proton transfer (ESIPT) or followed by photo-induced electron transfer (PET) lead to naked-eye color change, UV-vis spectral change, chemical shift in the NMR spectra and fluorescence spectral modifications. In this review we have tried to discuss about the anion sensing properties of Schiff base or reduced Schiff base receptors.

  11. Anion recognition by simple chromogenic and chromo-fluorogenic salicylidene Schiff base or reduced-Schiff base receptors

    NASA Astrophysics Data System (ADS)

    Dalapati, Sasanka; Jana, Sankar; Guchhait, Nikhil

    2014-08-01

    This review contains extensive application of anion sensing ability of salicylidene type Schiff bases and their reduced forms having various substituents with respect to phenolic sbnd OH group. Some of these molecular systems behave as receptor for recognition or sensing of various anions in organic or aqueous-organic binary solvent mixture as well as in the solid supported test kits. Development of Schiff base or reduced Schiff base receptors for anion recognition event is commonly based on the theory of hydrogen bonding interaction or deprotonation of phenolic -OH group. The process of charge transfer (CT) or inhibition of excited proton transfer (ESIPT) or followed by photo-induced electron transfer (PET) lead to naked-eye color change, UV-vis spectral change, chemical shift in the NMR spectra and fluorescence spectral modifications. In this review we have tried to discuss about the anion sensing properties of Schiff base or reduced Schiff base receptors.

  12. Expression profiling of pattern recognition receptors and selected cytokines in miniature dachshunds with inflammatory colorectal polyps.

    PubMed

    Igarashi, Hirotaka; Ohno, Koichi; Maeda, Shingo; Kanemoto, Hideyuki; Fukushima, Kenjiro; Uchida, Kazuyuki; Tsujimoto, Hajime

    2014-05-15

    Inflammatory colorectal polyps (ICRPs) are commonly seen in miniature dachshund (MD) dogs; typically, multiple polyps form with severe neutrophil infiltration. ICRP is thought to be a novel form of inflammatory bowel disease (IBD), but its etiology has not been investigated. The innate immune system is implicated in the pathogenesis of both human and canine IBD. Therefore, the aim of the current study was to evaluate the messenger RNA (mRNA) expression profiles of pattern recognition receptors (PRRs) and cytokines in ICRPs. Polyp tissues were collected by colonoscopic biopsies from 24 MDs with ICRPs. Non-polypoid colonic mucosa was collected from all MDs with ICRPs and 21 clinically healthy beagles (as the controls). The expression levels of the mRNAs encoding toll-like receptors (TLRs) 1-10; nucleotide-binding oligomerization domain (NOD)-like receptors NOD1 and NOD2; and cytokines IL-1β, IL-6, IL-8/CXCL8, and TNF-α were evaluated by quantitative real-time RT-PCR. Three of the 10 well-known candidate reference genes were selected as housekeeper genes based on analyses from the GeNorm, NormFinder, and BestKeeper programs. Levels of TLR1, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, NOD2, and all cytokines were significantly upregulated in the polyps relative to those in the controls. There was significant decrease in the expression levels of TLR3 and NOD1 in the polyp tissues compared to the non-polypoid colonic mucosa obtained from MDs with ICRPs. All upregulated PRR mRNAs were positively correlated with all proinflammatory cytokine mRNAs. This study demonstrated the dysregulation of PRRs and proinflammatory cytokines in ICRPs of MDs, which may play an important role in the pathogenesis of this disease.

  13. Intestinal microbiota diversity and expression of pattern recognition receptors in newly weaned piglets.

    PubMed

    Tao, Xin; Xu, Ziwei; Wan, Jing

    2015-04-01

    This study evaluated the gastrointestinal microbial diversity and the expression of pattern recognition receptors (PRRs) of the small intestine during the first week post-weaning in newly weaned piglets. Sixteen piglets were sacrificed on days 0, 1, 4, and 7 post-weaning. Luminal contents from the stomach, ileum, and colon were collected to determine the microbiota diversity; intestinal mucosa from the ileum was collected to assess mRNA expression of PRRs, including toll-like receptors (TLRs) and NOD-like receptors (NLRs); sections of ileum were examined immunohistochemically to assess the immunoglobulin-secreting cells. The results showed that the number of denaturing gradient gel electrophoresis (DGGE) bands from the ileum and colon contents were significantly reduced in the d 4 post-weaning group. Biodiversity indexes (Shannon-Wiener index, richness index, and evenness index) were significantly decreased in the ileum of weaning groups. These indexes decreased in the colon of the d 4 post-weaning group. No significant differences were obtained in the stomach. With the exception of TLR5, the mRNA expressions of TLR2, TLR4, and TLR7 increased post-weaning. The mRNA expressions of NOD1 and NOD2 were significantly affected in the d 4 post-weaning group, and there were no significant differences in the d 1 or d 7 post-weaning groups. Analysis of the immunoglobulin-secreting (IgA, IgG, and IgM) cells showed that the ratio of each immunoglobulin was significantly higher on d 7 than d 0. The results revealed that microbial diversity was lower in the ileum and on d 4 post-weaning. Weaning significantly affected the expression of intestinal PRRs mainly on d 1 and d 4 post-weaning. The expression of specific PRRs was triggered by weaning to recognize distinctive microbiota and promote the development and maturation of the intestinal mucosal immunity.

  14. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs

    PubMed Central

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-01-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an “Ω”-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

  15. Molecular Recognition of Agonist and Antagonist for Peroxisome Proliferator-Activated Receptor-α Studied by Molecular Dynamics Simulations

    PubMed Central

    Liu, Mengyuan; Wang, Lushan; Zhao, Xian; Sun, Xun

    2014-01-01

    Peroxisome proliferator activated receptor-α (PPAR-α) is a ligand-activated transcription factor which plays important roles in lipid and glucose metabolism. The aim of this work is to find residues which selectively recognize PPAR-α agonists and antagonists. To achieve this aim, PPAR-α/13M and PPAR-α/471 complexes were subjected to perform molecular dynamics simulations. This research suggests that several key residues only participate in agonist recognition, while some other key residues only contribute to antagonist recognition. It is hoped that such work is useful for medicinal chemists to design novel PPAR-α agonists and antagonists. PMID:24837836

  16. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.

    PubMed

    Calkin, Anna C; Goult, Benjamin T; Zhang, Li; Fairall, Louise; Hong, Cynthia; Schwabe, John W R; Tontonoz, Peter

    2011-12-13

    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

  17. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors

    PubMed Central

    Calkin, Anna C.; Goult, Benjamin T.; Zhang, Li; Fairall, Louise; Hong, Cynthia; Schwabe, John W. R.; Tontonoz, Peter

    2011-01-01

    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL–LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL–LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism. PMID:22109552

  18. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  19. Two Independent Histidines One in Human Prolactin and One in Its Receptor Are Critical for pH-dependent Receptor Recognition and Activation

    SciTech Connect

    M Kulkarni; M Tettamanzi; J Murphy; C Keeler; D Myszka; N Chayen; E Lolis; M Hodsdon

    2011-12-31

    Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL-receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL-receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

  20. Two Independent Histidines, One in Human Prolactin and One in Its Receptor, Are Critical for pH-dependent Receptor Recognition and Activation*

    PubMed Central

    Kulkarni, Mandar V.; Tettamanzi, M. Cristina; Murphy, James W.; Keeler, Camille; Myszka, David G.; Chayen, Naomi E.; Lolis, Elias J.; Hodsdon, Michael E.

    2010-01-01

    Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements. PMID:20889499

  1. Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

    PubMed Central

    Hong, Minsun; Lee, Peter S.; Hoffman, Ryan M. B.; Zhu, Xueyong; Krause, Jens C.; Laursen, Nick S.; Yoon, Sung-il; Song, Langzhou; Tussey, Lynda; Crowe, James E.; Ward, Andrew B.

    2013-01-01

    Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines. PMID:24027321

  2. An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Yi, Qilin; Song, Linsheng

    2016-01-01

    Accumulating evidence has demonstrated that leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and were involved in the immune response. In the present study, an LRR-only protein (designed as CfLRRop-1) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfLRRop-1 contained an open reading frame (ORF) of 1377 bp, which encoded a protein of 458 amino acids. An LRRNT motif, an LRR_7 motif and seven LRR motifs were found in the deduced amino acid sequence of CfLRRop-1. And these seven LRR motifs contained a conserved signature sequence LxxLxLxxNxL. The mRNA transcripts of CfLRRop-1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hepatopancreas. After the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), the mRNA transcripts of CfLRRop-1 in haemocytes all increased firstly within the first 6 h and secondly during 12-24 h post stimulation. The mRNA expression level of CfLRRop-1 was continuously up-regulated, after the expression of CfTLR (previously identified Toll-like receptor in C. farreri) was suppressed via RNA interference (RNAi). The recombinant CfLRRop-1 protein could directly bind LPS, PGN, GLU and poly I:C, and induce the release of TNF-α in mixed primary cultured scallop haemocytes. These results collectively indicated that CfLRRop-1 would function as a powerful pattern recognition receptor (PRR) and play a pivotal role in the immune response of scallops.

  3. Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site.

    PubMed

    Hong, Minsun; Lee, Peter S; Hoffman, Ryan M B; Zhu, Xueyong; Krause, Jens C; Laursen, Nick S; Yoon, Sung-Il; Song, Langzhou; Tussey, Lynda; Crowe, James E; Ward, Andrew B; Wilson, Ian A

    2013-11-01

    Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

  4. Functional analysis of pattern recognition receptors in miniature dachshunds with inflammatory colorectal polyps

    PubMed Central

    IGARASHI, Hirotaka; OHNO, Koichi; FUJIWARA-IGARASHI, Aki; KANEMOTO, Hideyuki; FUKUSHIMA, Kenjiro; GOTO-KOSHINO, Yuko; UCHIDA, Kazuyuki; TSUJIMOTO, Hajime

    2014-01-01

    Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan. MDs with ICRPs develop multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Therefore, ICRPs are thought to constitute a novel, breed-specific form of canine inflammatory bowel disease (IBD). Pattern recognition receptors (PRRs) play a key role in the distinction of pathogens from commensal bacteria and food antigens. Dysfunction resulting from genetic disorders of PRRs have been linked to human and canine IBD. Therefore, we analyzed the reactivity of PRRs in MDs with ICRPs. Twenty-six MDs with ICRPs and 16 control MDs were recruited. Peripheral blood-derived monocytes were obtained from each dog and then stimulated with PRR ligands for 6 and 24 hr; subsequently, messenger RNA (mRNA) expression levels and protein secretion of IL-1β were quantified using quantitative real-time PCR and ELISA, respectively. The levels of IL-1β mRNA and protein secretion after stimulation with a nucleotide-binding oligomerization domain 2 (NOD2) ligand were significantly greater in monocytes from ICRP-affected MDs than in those from control MDs. In addition, IL-1β protein secretion induced by toll-like receptor (TLR) 1/2, TLR2 and TLR2/6 stimulation was also significantly greater in ICRP-affected MDs. These results suggest that reactivity against NOD2, TLR1/2, TLR2 and TLR2/6 signals is enhanced in ICRP-affected MDs and may play a role in the pathogenesis of ICRPs in MDs. Additional studies of the genetic background of these PRRs should be performed. PMID:25650150

  5. Molecular recognition of amino acids with some fluorescent ditopic pyrylium- and pyridinium-based crown ether receptors

    NASA Astrophysics Data System (ADS)

    Moghimi, A.; Maddah, B.; Yari, A.; Shamsipur, M.; Boostani, M.; Fall Rastegar, M.; Ghaderi, A. R.

    2005-10-01

    The molecular recognition of L-amino acids such as asparagine, glutamine, lysine and arginine with some crownpyryliums, CP's, and a crownpyridinium compound, as receptors, were examined in methanol. 1H NMR spectroscopy was used to examine the structural stability of the receptors in the presence of the amino acids. The fluorimetric titration of the receptors by specified amino acids, other than arginine, was followed within a few minutes and the stoichiometry and stability of the resulting amino acid complexes were evaluated. The data analysis clearly demonstrated the critical role of the terminal amino group to carboxylic acid distance of amino acids for their proper fixation on the receptor molecules. Ion pairing for the two oppositely charged carboxylate anion and pyrylium (or pyridinium) cation, as well as the hydrogen bonding between crown ethers' oxygens and ammonium hydrogens are expected as the main interaction sources in the host-guest complexations.

  6. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens

    PubMed Central

    Lenzo, Jason C.; Holden, James A.; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T.; Yan, Yan; Caruso, Frank; Reynolds, Eric C.

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  7. Pattern recognition receptor-mediated cytokine response in infants across 4 continents⋆☆

    PubMed Central

    Smolen, Kinga K.; Ruck, Candice E.; Fortuno, Edgardo S.; Ho, Kevin; Dimitriu, Pedro; Mohn, William W.; Speert, David P.; Cooper, Philip J.; Esser, Monika; Goetghebuer, Tessa; Marchant, Arnaud; Kollmann, Tobias R.

    2014-01-01

    Background Susceptibility to infection as well as response to vaccination varies among populations. To date, the underlying mechanisms responsible for these clinical observations have not been fully delineated. Because innate immunity instructs adaptive immunity, we hypothesized that differences between populations in innate immune responses may represent a mechanistic link to variation in susceptibility to infection or response to vaccination. Objective Determine whether differences in innate immune responses exist among infants from different continents of the world. Methods We determined the innate cytokine response following pattern recognition receptor (PRR) stimulation of whole blood from 2-year-old infants across 4 continents (Africa, North America, South America, and Europe). Results We found that despite the many possible genetic and environmental exposure differences in infants across 4 continents, innate cytokine responses were similar for infants from North America, South America, and Europe. However, cells from South African infants secreted significantly lower levels of cytokines than did cells from infants from the 3 other sites, and did so following stimulation of extracellular and endosomal but not cytosolic PRRs. Conclusions Substantial differences in innate cytokine responses to PRR stimulation exist among different populations of infants that could not have been predicted. Delineating the underlying mechanism(s) for these differences will not only aid in improving vaccine-mediated protection but possibly also provide clues for the susceptibility to infection in different regions of the world. PMID:24290283

  8. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    PubMed

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.

  9. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    PubMed

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  10. Enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling.

    PubMed

    Harris, Katharine G; Coyne, Carolyn B

    2013-09-01

    Enteroviruses are the most common human viral pathogens worldwide. This genus of small, non-enveloped, single stranded RNA viruses includes coxsackievirus, rhinovirus, echovirus, and poliovirus species. Infection with these viruses can induce mild symptoms that resemble the common cold, but can also be associated with more severe syndromes such as poliomyelitis, neurological diseases including aseptic meningitis and encephalitis, myocarditis, and the onset of type I diabetes. In humans, polarized epithelial cells lining the respiratory and/or digestive tracts represent the initial sites of infection by enteroviruses. Control of infection in the host is initiated through the engagement of a variety of pattern recognition receptors (PRRs). PRRs act as the sentinels of the innate immune system and serve to alert the host to the presence of a viral invader. This review assembles the available data annotating the role of PRRs in the response to enteroviral infection as well as the myriad ways by which enteroviruses both interrupt and manipulate PRR signaling to enhance their own replication, thereby inducing human disease. PMID:23764548

  11. Biomimetic Taste Receptors with Chiral Recognition by Photoluminescent Metal-Organic Frameworks Chelated with Polyaniline Helices.

    PubMed

    Lee, Tu; Lin, Tsung Yan; Lee, Hung Lin; Chang, Yun Hsuan; Tsai, Yee Chen

    2016-01-22

    The adsorption of phenylaniline (Phe) enantiomers on (+)-polyaniline (PAN)-chelated [In(OH)(bdc)]n microcrystals was carefully designed and studied by using the Job titration, circular dichroism, X-ray photoelectron spectroscopy, and photoluminescence to mimic heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors in selective, but not specific, ligand binding with chiral recognition and signal transduction. Six essential working principles across different length scales are unraveled: 1) a chiral (+)-PAN (host), 2) specific sites for Phe-(+)/PAN (guest-host) binding, 3) a conformational change of (+)-PAN after binding with Phe enantiomers, 4) different degrees of packing for (+)-PAN, 5) interactions between (+)-PAN and the underlying signal-generating framework (i.e., [In(OH)(bdc)]n microcrystals), and 6) a systematic photoluminescent signal combination by using principal-component analysis from the other three polymer-chelated metal-organic frameworkds (MOFs), such as poly(acrylic acid) (PAA), sodium alginate (SA), and polyvinylpyrrolidone (PVP) to enhance the selectivity and discrimination capabilities.

  12. Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core.

    PubMed Central

    Glössl, J; Schubert-Prinz, R; Gregory, J D; Damle, S P; von Figura, K; Kresse, H

    1983-01-01

    Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues. Images Fig. 2. PMID:6316923

  13. Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4[alpha

    SciTech Connect

    Lu, Peng; Rha, Geun Bae; Melikishvili, Manana; Wu, Guangteng; Adkins, Brandon C.; Fried, Michael G.; Chi, Young-In

    2010-11-09

    HNF4{alpha} (hepatocyte nuclear factor 4{alpha}) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic {beta}-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4{alpha} is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4{alpha} recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 {angstrom} crystal structure of human HNF4{alpha} DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1{alpha}, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4{alpha} molecular function can cause significant effects in afflicted MODY patients.

  14. Systematic dissection of an aminopyrrolic cage receptor for β-glucopyranosides reveals the essentials for effective recognition.

    PubMed

    Francesconi, Oscar; Gentili, Matteo; Nativi, Cristina; Ardá, Ana; Cañada, F Javier; Jiménez-Barbero, Jesús; Roelens, Stefano

    2014-05-12

    A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor that selectively recognizes octyl-β-D-glucopyranoside (OctβGlc). NMR spectroscopy and isothermal titration calorimetry binding measurements showed that cleavage of one pillar of the cage was beneficial to the binding properties of the receptor, as long as two residual amino groups of the cleaved pillar were present. Removal of these two residual amino groups produced a dramatic loss of affinity for OctβGlc of the resulting monocyclic analogue of the parent cage receptor. A significant improvement in the binding ability was achieved by replacing one pillar with two aminopyrrolic hydrogen-bonding arms, despite the loss of a preorganized structure. In contrast to the cage receptor, recognition of OctβGlc was observed, even in a competitive medium (30 % DMF in chloroform). Structural studies in solution, carried out through NMR spectroscopy and molecular modeling calculations, led to the elucidation of the 3D binding modes of the side-armed monocyclic receptors; this highlighted the key role of the amino groups and demonstrated the occurrence of a rotaxane-like complex, which featured the octyl chain of the glucoside threaded through the macrocyclic ring.

  15. Structural characteristics of the recognition site for cholinergic ligands in the nicotinic acetylcholine receptor from squid optical ganglia

    SciTech Connect

    Plyashkevich, Yu.G.; Demushkin, V.P.

    1986-01-20

    The influence of chemical modification on the parameters of the binding of cholinergic ligands by the nicotinic acetylcholine receptor of squid optical ganglia was investigated. The presence of two subpopulations of recognition sites, differing in the composition of the groups contained in them, was detected. It was established with high probability that subpopulation I contains arginine and tyrosine residues and a carboxyl group while subpopulation II contains an amino group, a thyrosine residue, and a carboxyl group. Moreover, in both subpopulations there is an amino group important only for the binding of tubocurarin. On the basis of the results obtained, a model of the recognition sites for cholinergic ligands of the nicotinic acetylcholine receptor of squid optical ganglia is proposed.

  16. NLRP7 and related inflammasome activating pattern recognition receptors and their function in host defense and disease.

    PubMed

    Radian, Alexander D; de Almeida, Lucia; Dorfleutner, Andrea; Stehlik, Christian

    2013-01-01

    Host defense requires the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and the induction of pyroptotic cell death, which depends on the activation of inflammatory Caspases within inflammasomes by innate immune cells. Several cytosolic pattern recognition receptors (PRRs) have been implicated in this process in response to infectious and sterile agonists. Here we summarize the current knowledge on inflammasome-organizing PRRs, emphasizing the recently described NLRP7, and their implications in human disease.

  17. The broad pattern recognition spectrum of the Toll-like receptor in mollusk Zhikong scallop Chlamys farreri.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Sun, Rui; Yue, Feng; Yi, Qilin; Song, Linsheng

    2015-10-01

    Toll-like receptors (TLRs) are among the most studied pattern recognition receptors (PRRs) playing essential roles in innate immune defenses. In the present study, the basic features of CfTLR in mollusk Zhikong scallop Chlamys farreri, including sequence homology, tissue distribution, subcellular localization and ligands spectrum, were investigated to elucidate its pattern recognition. The elements of extracellular domains (ECD) in CfTLR displayed high homology to the corresponding parts of the ECDs in TLRs from Homo sapiens. CfTLR protein was detected in hemocytes, mantle, gills, hepatopancreas, kidney and gonad of the scallops, and it was localized in both the plasma membranes and the lysosomes in HEK293T cells. CfTLR could activate NFκB in response to multiple HsTLR ligands including Pam3CSK4, glucan (GLU), peptidoglycan (PGN), polyriboinosinic:polyribocytidylic acid (poly I:C), Imiquimod and three types of CpG. Additionally, the scallop serum could enhance the induction of NFκB in the CfTLR expressing cells elicited by most PAMPs, including GLU, PGN, Imiquimod and four types of CpG. It could be concluded that this primitive mollusk TLR shared a hybrid function in pattern recognition and could recognize broader ligands than mammalian TLRs, and its mosaic capability of pathogen associated molecular pattern (PAMP) recognition might be based on the basic features of its structure, ligand properties and the assistance of some components in scallop serum.

  18. Structural basis of RNA recognition and activation by innate immune receptor RIG-I

    SciTech Connect

    Jiang, Fuguo; Ramanathan, Anand; Miller, Matthew T.; Tang, Guo-Qing; Gale, Jr., Michael; Patel, Smita S.; Marcotrigiano, Joseph

    2012-05-29

    Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA

  19. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

    PubMed Central

    Haberland, M E; Fogelman, A M

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor. PMID:3857610

  20. Matricryptic sites control tissue injury responses in the cardiovascular system: relationships to pattern recognition receptor regulated events.

    PubMed

    Davis, George E

    2010-03-01

    This review addresses new concepts related to the importance of how cells within the cardiovascular system respond to matricryptic sites generated from the extracellular matrix (ECM) following tissue injury. A model is presented whereby matricryptic sites exposed from the ECM result in activation of multiple cell surface receptors including integrins, scavenger receptors, and toll-like receptors which together are hypothesized to coactivate downstream signaling pathways which alter cell behaviors following tissue injury. Of great interest are the relationships between matricryptic fragments of ECM called matricryptins and other stimuli that activate cells during injury states such as released components from cells (DNA, RNA, cytoskeletal components such as actin) or products from infectious agents in innate immunity responses. These types of cell activating molecules, which are composed of repeating molecular elements, are known to interact with pattern recognition receptors that (i) are expressed from cell surfaces, (ii) are released from cells following tissue injury, or (iii) circulate as components of plasma. Thus, cell recognition of matricryptic sites from the ECM appears to be an important component of a broad cell and tissue sensory system to detect and respond to environmental cues generated following varied types of tissue injury.

  1. Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis.

    PubMed

    Qin, Liang; Guimarães, Dimitrius Santiago P S F; Melesse, Michael; Hall, Mark C

    2016-07-22

    The anaphase-promoting complex, or cyclosome (APC/C), is a ubiquitin ligase that selectively targets proteins for degradation in mitosis and the G1 phase and is an important component of the eukaryotic cell cycle control system. How the APC/C specifically recognizes its substrates is not fully understood. Although well characterized degron motifs such as the destruction box (D-box) and KEN-box are commonly found in APC/C substrates, many substrates apparently lack these motifs. A variety of alternative APC/C degrons have been reported, suggesting either that multiple modes of substrate recognition are possible or that our definitions of degron structure are incomplete. We used an in vivo yeast assay to compare the G1 degradation rate of 15 known substrates of the APC/C co-activator Cdh1 under normal conditions and conditions that impair binding of D-box, KEN-box, and the recently identified ABBA motif degrons to Cdh1. The D-box receptor was required for efficient proteolysis of all Cdh1 substrates, despite the absence of canonical D-boxes in many. In contrast, the KEN-box receptor was only required for normal proteolysis of a subset of substrates and the ABBA motif receptor for a single substrate in our system. Our results suggest that binding to the D-box receptor may be a shared requirement for recognition and processing of all Cdh1 substrates.

  2. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor. PMID:27417229

  3. Self-regulation and cross-regulation of pattern-recognition receptor signalling in health and disease.

    PubMed

    Cao, Xuetao

    2016-01-01

    In the initiation of innate immune responses against pathogens, pattern-recognition receptors (PRRs) have an essential role in recognizing specific components of microorganisms and triggering responses that eliminate the invading microorganisms. However, inappropriate activation of PRRs can lead to prolonged inflammation and even to autoimmune and inflammatory diseases. Thus, PRR-triggered responses are regulated through the degradation or translocation of the innate receptors themselves and through the involvement of intracellular regulators or amplifiers. In addition, a complex interplay between PRRs and/or other immune pathways finely tunes the outcome of host immune defence responses. In this Review, I describe many of the numerous distinct mechanisms for the self-regulation and cross-regulation of innate immune receptor signalling.

  4. Expression of pathogen recognition receptors and pro-inflammatory cytokine transcripts in clinical and sub-clinical endometritis cows.

    PubMed

    Loyi, Tumnyak; Kumar, Harendra; Nandi, Sukdeb; Patra, Manas Kumar

    2015-01-01

    The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with

  5. Expression of pathogen recognition receptors and pro-inflammatory cytokine transcripts in clinical and sub-clinical endometritis cows.

    PubMed

    Loyi, Tumnyak; Kumar, Harendra; Nandi, Sukdeb; Patra, Manas Kumar

    2015-01-01

    The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with

  6. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    PubMed Central

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  7. Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

    PubMed

    Kohler, Amanda C; Chen, Li-Hung; Hurlburt, Nicholas; Salvucci, Anthony; Schwessinger, Benjamin; Fisher, Andrew J; Stergiopoulos, Ioannis

    2016-08-01

    Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector. PMID:27401545

  8. Differential Involvement of Dopamine D1 Receptor and MEK Signaling Pathway in the Ventromedial Prefrontal Cortex in Consolidation and Reconsolidation of Recognition Memory

    ERIC Educational Resources Information Center

    Maroun, Mouna; Akirav, Irit

    2009-01-01

    We investigated MEK and D1 receptors in the ventromedial prefrontal cortex (vmPFC) in consolidation and reconsolidation of recognition memory in rats nonhabituated to the experimental context (NH) or with reduced arousal due to extensive prior habituation (H). The D1 receptor antagonist enhanced consolidation and impaired reconsolidation in NH but…

  9. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  10. Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

    SciTech Connect

    Albright, Seth; Chen Bin; Holbrook, Kristen; Jain, Nitin U.

    2008-04-04

    CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.

  11. The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules.

    PubMed

    Le Saux, Agnès; Ng, Patricia Miang Lon; Koh, Joanne Jing Yun; Low, Diana Hooi Ping; Leong, Geraldine E-Ling; Ho, Bow; Ding, Jeak Ling

    2008-03-28

    Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core "pathogen-recognition complex" via their complement control protein modules. PMID:18279891

  12. Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2.

    PubMed

    Schmiedeberg, Kristin; Shirokova, Elena; Weber, Hans-Peter; Schilling, Boris; Meyerhof, Wolfgang; Krautwurst, Dietmar

    2007-09-01

    An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.

  13. The changing of the guard: the Pto/Prf receptor complex of tomato and pathogen recognition.

    PubMed

    Ntoukakis, Vardis; Saur, Isabel M L; Conlan, Brendon; Rathjen, John P

    2014-08-01

    One important model for disease resistance is the Prf recognition complex of tomato, which responds to different bacterial effectors. Prf incorporates a protein kinase called Pto as its recognition domain that mimics effector virulence targets, and activates resistance after interaction with specific effectors. Recent findings show that this complex is oligomeric, and reveal how this impacts mechanism. Oligomerisation brings two or more kinases into proximity, where they can phosphorylate each other after effector perception. Effector attack on one kinase activates another in trans, constituting a molecular trap for the effector. Oligomerisation of plant resistance proteins may be a general concept that broadens pathogen recognition and restricts the ability of pathogens to evolve virulence.

  14. Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

    PubMed Central

    Modun, Belinda; Evans, Robert W.; Joannou, Christopher L.; Williams, Paul

    1998-01-01

    provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin. PMID:9673237

  15. Posttraining activation of CB1 cannabinoid receptors in the CA1 region of the dorsal hippocampus impairs object recognition long-term memory.

    PubMed

    Clarke, Julia R; Rossato, Janine I; Monteiro, Siomara; Bevilaqua, Lia R M; Izquierdo, Iván; Cammarota, Martín

    2008-09-01

    Evidence indicates that brain endocannabinoids are involved in memory processing. However, the participation of CB1 and CB2 cannabinoid receptors in recognition memory has not been yet conclusively determined. Therefore, we evaluated the effect of the posttraining activation of hippocampal cannabinoid receptors on the consolidation of object recognition memory. Rats with infusion cannulae stereotaxically aimed to the CA1 region of the dorsal hippocampus were trained in an object recognition learning task involving exposure to two different stimulus objects. Memory retention was assessed at different times after training. In the test sessions, one of the objects presented during training was replaced by a novel one. When infused in the CA1 region immediately after training, the non-selective cannabinoid receptor agonist WIN-55,212-2 and the endocannabinoid membrane transporter inhibitor VDM-11 blocked long-term memory retention in a dose-dependent manner without affecting short-term memory, exploratory behavior, anxiety state or the functionality of the hippocampus. The amnesic effect of WIN-55,212-2 and VDM-11 was not due to state-dependency and was completely reversed by co-infusion of the CB1 receptor antagonist AM-251 and mimicked by the CB1 receptor agonist ACEA but not by the CB2 receptor agonists JWH-015 and palmitoylethanolamide. Our data indicate that activation of hippocampal CB1 receptors early after training hampers consolidation of object recognition memory.

  16. First identification of Toll-like receptor-4 in avian brain: evolution of lipopolysaccharide recognition and inflammation-dependent responses.

    PubMed

    Saponaro, Concetta; Cianciulli, Antonia; Calvello, Rosa; Cavallo, Pasqua; Mitolo, Vincenzo; Panaro, Maria Antonietta

    2011-03-01

    In this work, we examine the effects of lipopolysaccharide (LPS) treatment on nerve cells of chick embryo used as a universal avian model. We demonstrate that LPS leads to a dramatic cell loss in primary cultures of both glia and neurons, isolated from chick embryos. Toxic effects appear to be mediated by the Toll-like receptor (TLR)-4 complex, expressed in both glial and neuronal cells, since after TLR-4 silencing by RNA interference experiments LPS-induced cytotoxicity was prevented. The role of nitric oxide in LPS-induced cell damage has also been investigated. These results demonstrate, for the first time in avian nerve cells, the surface expression of TLR-4 and its role as a pattern recognition receptor involved in LPS-induced cell responses in a similar manner to that observed in mammals.

  17. Structure of the Neisserial outer membrane protein Opa₆₀: loop flexibility essential to receptor recognition and bacterial engulfment.

    PubMed

    Fox, Daniel A; Larsson, Per; Lo, Ryan H; Kroncke, Brett M; Kasson, Peter M; Columbus, Linda

    2014-07-16

    The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

  18. Structure of the Neisserial outer membrane protein Opa₆₀: loop flexibility essential to receptor recognition and bacterial engulfment.

    PubMed

    Fox, Daniel A; Larsson, Per; Lo, Ryan H; Kroncke, Brett M; Kasson, Peter M; Columbus, Linda

    2014-07-16

    The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors. PMID:24813921

  19. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles.

    PubMed

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level.

  20. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles.

    PubMed

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level. PMID:26449412

  1. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles

    PubMed Central

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level. PMID:26449412

  2. Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants

    PubMed Central

    Walker, Robin K.; Zhao, Yichen; Berkowitz, Gerald A.

    2012-01-01

    Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca2+ elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca2+ elevation is attributable to cGMP activation of a Ca2+ channel. Some differences were identified between Pep/PEPR signaling and the Ca2+-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca2+ stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca2+. Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca2+ elevation, H2O2 generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen. PMID:23150556

  3. Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants.

    PubMed

    Ma, Yi; Walker, Robin K; Zhao, Yichen; Berkowitz, Gerald A

    2012-11-27

    Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca(2+) elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca(2+) elevation is attributable to cGMP activation of a Ca(2+) channel. Some differences were identified between Pep/PEPR signaling and the Ca(2+)-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca(2+) stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca(2+). Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca(2+) elevation, H(2)O(2) generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen.

  4. CRIg Functions as a Macrophage Pattern Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

    PubMed

    Zeng, Zhutian; Surewaard, Bas G J; Wong, Connie H Y; Geoghegan, Joan A; Jenne, Craig N; Kubes, Paul

    2016-07-13

    Kupffer cells (KCs), the vast pool of intravascular macrophages in the liver, help to clear blood-borne pathogens. The mechanisms by which KCs capture circulating pathogens remain unknown. Here we use intra-vital imaging of mice infected with Staphylococcus aureus to directly visualize the dynamic process of bacterial capture in the liver. Circulating S. aureus were captured by KCs in a manner dependent on the macrophage complement receptor CRIg, but the process was independent of complement. CRIg bound Staphylococcus aureus specifically through recognition of lipoteichoic acid (LTA), but not cell-wall-anchored surface proteins or peptidoglycan. Blocking the recognition between CRIg and LTA in vivo diminished the bacterial capture in liver and led to systemic bacterial dissemination. All tested Gram-positive, but not Gram-negative, bacteria bound CRIg in a complement-independent manner. These findings reveal a pattern recognition role for CRIg in the direct capture of circulating Gram-positive bacteria from the bloodstream. PMID:27345697

  5. Structure of an LDLR-RAP Complex Reveals a General Mode for Ligand Recognition by Lipoprotein Receptors

    SciTech Connect

    Fisher,C.; Beglova, N.; Blacklow, s.

    2006-01-01

    Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 Angstroms X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.

  6. Selectivity of pyoverdine recognition by the FpvA receptor of Pseudomonas aeruginosa from molecular dynamics simulations.

    PubMed

    Bouvier, Benjamin; Cézard, Christine; Sonnet, Pascal

    2015-07-21

    The Gram-negative bacterium Pseudomonas aeruginosa, a ubiquitous human opportunistic pathogen, has developed resistances to multiple antibiotics. It uses its primary native siderophore, pyoverdine, to scavenge the iron essential to its growth in the outside medium and transport it back into its cytoplasm. The FpvA receptor on the bacterial outer membrane recognizes and internalizes pyoverdine bearing its iron payload, but can also bind pyoverdines from other Pseudomonads or synthetic analogues. Pyoverdine derivatives could therefore be used as vectors to deliver antibiotics into the bacterium. In this study, we use molecular dynamics and free energy calculations to characterize the mechanisms and thermodynamics of the recognition of the native pyoverdines of P. aeruginosa and P. fluorescens by FpvA. Based on these results, we delineate the features that pyoverdines with high affinity for FpvA should possess. In particular, we show that (i) the dynamics and interaction of the unbound pyoverdines with water should be optimized with equal care as the interface contacts in the complex with FpvA; (ii) the C-terminal extremity of the pyoverdine chain, which appears to play no role in the bound complex, is involved in the intermediate stages of recognition; and (iii) the length and cyclicity of the pyoverdine chain can be used to fine-tune the kinetics of the recognition mechanism. PMID:26098682

  7. Stereoselective recognition of the enantiomers of phenglutarimide and of six related compounds by four muscarinic receptor subtypes.

    PubMed Central

    Waelbroeck, M.; Lazareno, S.; Pfaff, O.; Friebe, T.; Tastenoy, M.; Mutschler, E.; Lambrecht, G.

    1996-01-01

    1. We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M1 receptors in NB-OK-1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2) and guinea-pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human m1-m4 receptors expressed by CHO cells, using [3H]-N-methylscopolamine ([3H]-NMS) as radioligand. 2. The high affinity enantiomers behaved as competitive antagonists in binding and pharmacological studies. (S)-phenglutarimide (pKi-M1 = 9.0/9.3) and (R)-thienglutarimide (pKi-M1 = 8.6/9.2) recognized selectively the native M1 > M4 > M3 > M2 receptors in tissues as well as the respective cloned receptors. 3. The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea-pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as M1/M4-like, M2 and M3, respectively. 4. Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine-2,6-dione nitrogen also reduced markedly the eutomers' affinities, more on the M1 than on the other subtypes. 5. The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)-thienglutarimide used in this study was contaminated by less than

  8. Vγ2Vδ2 T Cell Receptor Recognition of Prenyl Pyrophosphates is Dependent on all Complementarity Determining Regions1

    PubMed Central

    Wang, Hong; Fang, Zhimei; Morita, Craig T.

    2010-01-01

    γδ T cells differ from αβ T cells in the antigens they recognize and their functions in immunity. While most αβ T cell receptors (TCR) recognize peptides presented by MHC class I or II, human γδ T cells expressing Vγ2Vδ2 TCRs recognize nonpeptide prenyl pyrophosphates. To define the molecular basis for this recognition, the effect of mutations in the TCR complementarity-determining regions (CDR) was assessed. Mutations in all CDR loops altered recognition and cover a large footprint. Unlike murine γδ TCR recognition of the MHC class Ib T22 protein, there was no CDR3δ motif required for recognition because only 1 residue is required. Instead, the length and sequence of CDR3γ was key. Although a potential prenyl pyrophosphate-binding site was defined by Lys109 in Jγ1.2 and Arg51 in CDR2δ, the area outlined by critical mutations is much larger. These results show that prenyl pyrophosphate recognition is primarily by germline-encoded regions of the γδ TCR, allowing a high proportion of Vγ2Vδ2 TCRs to respond. This underscores its parallels to innate immune receptors. Our results also provide strong evidence for the existence of an antigen-presenting molecule for prenyl pyrophosphates. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org. PMID:20483784

  9. Structural basis for molecular recognition of folic acid by folate receptors.

    PubMed

    Chen, Chen; Ke, Jiyuan; Zhou, X Edward; Yi, Wei; Brunzelle, Joseph S; Li, Jun; Yong, Eu-Leong; Xu, H Eric; Melcher, Karsten

    2013-08-22

    Folate receptors (FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate cellular uptake of folate. Although expressed at very low levels in most tissues, folate receptors, especially FRα, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions. The folate dependency of many tumours has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, high-affinity antifolates, folate-based imaging agents and folate-conjugated drugs and toxins. To understand how folate binds its receptors, we determined the crystal structure of human FRα in complex with folic acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulphide bonds and contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for designing more specific drugs targeting the folate receptor system.

  10. Effects of the nicotinic α7 receptor partial agonist GTS-21 on NMDA-glutamatergic receptor related deficits in sensorimotor gating and recognition memory in rats

    PubMed Central

    Terry, Alvin V.; Tehim, Ashok

    2016-01-01

    Rationale Disturbances in information processing and cognitive function are key features of schizophrenia. Nicotinic α7 acetylcholine receptors (α7-nAChR) are involved in sensory gating and cognition, thereby representing a viable therapeutic strategy. Objectives and methods We investigated the effects of GTS-21, an α7-nAChR partial agonist, on prepulse inhibition (PPI) of acoustic startle in two pharmacologic impairment models in Wistar male rats: NMDA-glutamate receptor antagonism by MK-801 and dopamine receptor agonism by apomorphine. The cognitive effects of GTS-21 were assessed using the object recognition task (ORT) at short (3 h) and long (48 h) delays in Sprague-Dawley male rats. Pharmacological specificity was assessed by methyllycaconitine (MLA) coadministration with GTS-21. Results In the PPI task, GTS-21 (1–10 mg/kg) alone did not alter the PPI response or startle amplitude. Coadministration of GTS-21 with MK-801 (0.1 mg/kg) or apomorphine (0.5 mg/kg) abolished the pharmacologic-induced PPI impairment as did the antipsychotics clozapine (5.0 mg/kg) and haloperidol (0.3 mg/kg). MK-801 alone increased startle amplitude which was blocked by GTS-21. In the ORT, GTS-21 (0.1–10 mg/kg) reversed the MK-801 (0.08 mg/kg)-induced memory deficit at the 3 h delay and enhanced memory at the 48 h delay, an effect abolished by MLA (0.313–5 mg/kg). Conclusions The results extend our preclinical pharmacological understanding of GTS-21 to include the ability of GTS-21 to modulate NMDA-glutamate receptor function, in vivo. Given the role of NMDA-glutamate receptor involvement in schizophrenia, α7-nAChR agonists may represent a novel treatment strategy for the pathophysiological deficits of schizophrenia and other psychiatric disorders. PMID:24595504

  11. Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVβ20-1, altering pHLA recognition.

    PubMed

    Watkins, Stephan; Pichler, Werner J

    2013-01-01

    T cell receptors (TCR) containing Vβ20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vβ20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2β domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vβ20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable β or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2β loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2β loop amino acid positions. These residues, TYRβ57, ASPβ64, and LYSβ65 initially hold hydrogen bonds from the CDR2β to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2β of Vβ20-1 acts as a ligand controlled switch affecting overall TCR

  12. Dopamine receptor D4 (DRD4) gene modulates the influence of informational masking on speech recognition.

    PubMed

    Xie, Zilong; Maddox, W Todd; Knopik, Valerie S; McGeary, John E; Chandrasekaran, Bharath

    2015-01-01

    Listeners vary substantially in their ability to recognize speech in noisy environments. Here we examined the role of genetic variation on individual differences in speech recognition in various noise backgrounds. Background noise typically varies in the levels of energetic masking (EM) and informational masking (IM) imposed on target speech. Relative to EM, release from IM is hypothesized to place greater demand on executive function to selectively attend to target speech while ignoring competing noises. Recent evidence suggests that the long allele variant in exon III of the DRD4 gene, primarily expressed in the prefrontal cortex, may be associated with enhanced selective attention to goal-relevant high-priority information even in the face of interference. We investigated the extent to which this polymorphism is associated with speech recognition in IM and EM conditions. In an unscreened adult sample (Experiment 1) and a larger screened replication sample (Experiment 2), we demonstrate that individuals with the DRD4 long variant show better recognition performance in noise conditions involving significant IM, but not in EM conditions. In Experiment 2, we also obtained neuropsychological measures to assess the underlying mechanisms. Mediation analysis revealed that this listening condition-specific advantage was mediated by enhanced executive attention/working memory capacity in individuals with the long allele variant. These findings suggest that DRD4 may contribute specifically to individual differences in speech recognition ability in noise conditions that place demands on executive function. PMID:25497692

  13. Dopamine receptor D4 (DRD4) gene modulates the influence of informational masking on speech recognition

    PubMed Central

    Xie, Zilong; Maddox, W. Todd; Knopik, Valerie S.; McGeary, John E.; Chandrasekaran, Bharath

    2014-01-01

    Listeners vary substantially in their ability to recognize speech in noisy environments. Here we examined the role of genetic variation on individual differences in speech recognition in various noise backgrounds. Background noise typically varies in the levels of energetic masking (EM) and informational masking (IM) imposed on target speech. Relative to EM, release from IM is hypothesized to place greater demand on executive function to selectively attend to target speech while ignoring competing noises. Recent evidence suggests that the long allele variant in exon III of the DRD4 gene, primarily expressed in the prefrontal cortex, may be associated with enhanced selective attention to goal-relevant high-priority information even in the face of interference. We investigated the extent to which this polymorphism is associated with speech recognition in IM and EM conditions. In an unscreened adult sample (Experiment 1) and a larger screened replication sample (Experiment 2), we demonstrate that individuals with the DRD4 long variant show better recognition performance in noise conditions involving significant IM, but not in EM conditions. In Experiment 2, we also obtained neuropsychological measures to assess the underlying mechanisms. Mediation analyses revealed that this listening condition-specific advantage was mediated by enhanced executive attention/working memory capacity in individuals with the long allele variant. These findings suggest that DRD4 may contribute specifically to individual differences in speech recognition ability in noise conditions that place demands on executive function. PMID:25497692

  14. Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.

    PubMed

    Labéta, M O; Vidal, K; Nores, J E; Arias, M; Vita, N; Morgan, B P; Guillemot, J C; Loyaux, D; Ferrara, P; Schmid, D; Affolter, M; Borysiewicz, L K; Donnet-Hughes, A; Schiffrin, E J

    2000-05-15

    Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

  15. Recognition of a Functional Peroxisome Type 1 Target by the Dynamic Import Receptor Pex5p

    PubMed Central

    Stanley, Will A.; Filipp, Fabian V.; Kursula, Petri; Schüller, Nicole; Erdmann, Ralf; Schliebs, Wolfgang; Sattler, Michael; Wilmanns, Matthias

    2016-01-01

    Summary Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function. PMID:17157249

  16. Supramolecular Ensembles Formed between Charged Conjugated Polymers and Glycoprobes for the Fluorogenic Recognition of Receptor Proteins.

    PubMed

    Dou, Wei-Tao; Zeng, Ya-Li; Lv, Ying; Wu, Jiatao; He, Xiao-Peng; Chen, Guo-Rong; Tan, Chunyan

    2016-06-01

    This paper describes the simple construction of a unique class of supramolecular ensembles formed by electrostatic self-assembly between charged conjugated polymers and fluorophore-coupled glycoligands (glycoprobes) for the selective fluorogenic detection of receptor proteins at both the molecular and cellular levels. We show that positively and negatively charged diazobenzene-containing poly(p-phenylethynylenes) (PPEs) can be used to form stable fluorogenic probes with fluorescein-based (negatively charged) and rhodamine B based (positively charged) glycoprobes by electrostatic interaction. The structures of the ensembles have been characterized by spectroscopic and microscopic techniques. The supramolecular probes formed show quenched fluorescence in an aqueous buffer solution, which can be specifically recovered, in a concentration-dependent manner, through competitive complexation with a selective protein receptor, over a range of other unselective proteins. The ensembles also show selective fluorescence enhancement with a live cell that expresses the glycoligand receptor but not a control cell without receptor expression. PMID:27159586

  17. Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1

    PubMed Central

    Chen, Guo-Yun; Brown, Nicholas K; Wu, Wei; Khedri, Zahra; Yu, Hai; Chen, Xi; van de Vlekkert, Diantha; D'Azzo, Alessandra; Zheng, Pan; Liu, Yang

    2014-01-01

    Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection. DOI: http://dx.doi.org/10.7554/eLife.04066.001 PMID:25187624

  18. Structures of the Signal Recognition Particle Receptor From the Archaeon Pyrococcus Furiosus: Implications for the Targeting Step at the Membrane

    SciTech Connect

    Egea, P.F.; Tsuruta, H.; Leon, G.P.de; Napetschnig, J.; Walter, P.; Stroud, R.M.

    2009-05-18

    In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP {center_dot} magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP {center_dot} SR targeting complexes.

  19. Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1.

    PubMed

    Chen, Guo-Yun; Brown, Nicholas K; Wu, Wei; Khedri, Zahra; Yu, Hai; Chen, Xi; van de Vlekkert, Diantha; D'Azzo, Alessandra; Zheng, Pan; Liu, Yang

    2014-09-03

    Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.

  20. Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

    PubMed Central

    Egea, Pascal F.; Tsuruta, Hiro; de Leon, Gladys P.; Napetschnig, Johanna; Walter, Peter; Stroud, Robert M.

    2008-01-01

    In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes. PMID:18978942

  1. Ebola virus entry requires the host-programmed recognition of an intracellular receptor.

    PubMed

    Miller, Emily Happy; Obernosterer, Gregor; Raaben, Matthijs; Herbert, Andrew S; Deffieu, Maika S; Krishnan, Anuja; Ndungo, Esther; Sandesara, Rohini G; Carette, Jan E; Kuehne, Ana I; Ruthel, Gordon; Pfeffer, Suzanne R; Dye, John M; Whelan, Sean P; Brummelkamp, Thijn R; Chandran, Kartik

    2012-04-18

    Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane. PMID:22395071

  2. Danger- and pathogen-associated molecular patterns recognition by pattern-recognition receptors and ion channels of the transient receptor potential family triggers the inflammasome activation in immune cells and sensory neurons.

    PubMed

    Santoni, Giorgio; Cardinali, Claudio; Morelli, Maria Beatrice; Santoni, Matteo; Nabissi, Massimo; Amantini, Consuelo

    2015-02-03

    An increasing number of studies show that the activation of the innate immune system and inflammatory mechanisms play an important role in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms and its activation occurs in response to pathogens or tissue injury via pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Intracellular pathways, linking immune and inflammatory response to ion channel expression and function, have been recently identified. Among ion channels, the transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge of interactions between immune cells and PRRs and ion channels of TRP families with PAMPs and DAMPs to provide new insights into the pathogenesis of inflammatory diseases. TRP channels have been found to interfere with innate immunity via both nuclear factor-kB and procaspase-1 activation to generate the mature caspase-1 that cleaves pro-interleukin-1β cytokine into the mature interleukin-1β.Sensory neurons are also adapted to recognize dangers by virtue of their sensitivity to intense mechanical, thermal and irritant chemical stimuli. As immune cells, they possess many of the same molecular recognition pathways for danger. Thus, they express PRRs including Toll-like receptors 3, 4, 7, and 9, and stimulation by Toll-like receptor ligands leads to induction of inward currents and sensitization in TRPs. In addition, the expression of inflammasomes in neurons and the involvement of TRPs in central nervous system diseases strongly support a role of TRPs in inflammasome-mediated neurodegenerative pathologies. This field is still at its beginning and further studies may be required.Overall, these

  3. Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

    PubMed Central

    Barnes, J. M.; Barnes, N. M.; Costall, B.; Jagger, S. M.; Naylor, R. J.; Robertson, D. W.; Roe, S. Y.

    1992-01-01

    1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1559139

  4. Transcriptome Profiling Reveals Higher Vertebrate Orthologous of Intra-Cytoplasmic Pattern Recognition Receptors in Grey Bamboo Shark

    PubMed Central

    Gupta, Ravi; Gopal, Dhinakar Raj; Rajesh, Preeti; Chidambaram, Balachandran; Kalyanasundaram, Aravindan; Angamuthu, Raja

    2014-01-01

    From an immunologist perspective, sharks are an important group of jawed cartilaginous fishes and survey of the public database revealed a great gap in availability of large-scale sequence data for the group of Chondrichthyans the elasmobranchs. In an attempt to bridge this deficit we generated the transcriptome from the spleen and kidney tissues (a total of 1,606,172 transcripts) of the shark, Chiloscyllium griseum using the Illumina HiSeq2000 platform. With a cut off of > = 300 bp and an expression value of >1RPKM we used 43,385 transcripts for BLASTX analysis which revealed 17,548 transcripts matching to the NCBI nr database with an E-value of < = 10−5 and similarity score of 40%. The longest transcript was 16,974 bases with matched to HECT domain containing E3 ubiqutin protein ligase. MEGAN4 annotation pipeline revealed immune and signalling pathways including cell adhesion molecules, cytokine-cytokine receptor interaction, T-cell receptor signalling pathway and chemokine signaling pathway to be highly expressed in spleen, while different metabolism pathways such as amino acid metabolism, carbohydrate metabolism, lipid metabolism and xenobiotic biodegradation were highly expressed in kidney. Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction. We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway. The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs. PMID:24956167

  5. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

    NASA Astrophysics Data System (ADS)

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M.; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

  6. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors.

    PubMed

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S; Stojanovic, Milan N

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects. PMID:25343606

  7. Recognition and Sensing of Low-Epitope Targets via Ternary Complexes with Oligonucleotides and Synthetic Receptors

    PubMed Central

    Yang, Kyung-Ae; Barbu, Michaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2015-01-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and selectivity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose, or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a non-specific organometallic receptor. The method is general and enables broad clinical use of aptamers for detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects. PMID:25343606

  8. Buggy Creek virus (Togaviridae: Alphavirus) upregulates expression of pattern recognition receptors and interferons in House Sparrows (Passer domesticus).

    PubMed

    Fassbinder-Orth, Carol A; Barak, Virginia A; Rainwater, Ellecia L; Altrichter, Ashley M

    2014-06-01

    Birds serve as reservoirs for at least 10 arthropod-borne viruses, yet specific immune responses of birds to arboviral infections are relatively unknown. Here, adult House Sparrows were inoculated with an arboviral alphavirus, Buggy Creek virus (BCRV), or saline, and euthanized between 1 and 3 days postinoculation. Virological dynamics and gene expression dynamics were investigated. Birds did not develop viremia postinoculation, but cytopathic virus was found in the skeletal muscle and spleen of birds 1 and 3 days postinoculation (DPI). Viral RNA was detected in the blood of BCRV-infected birds 1 and 2 DPI, in oral swabs 1-3 DPI, and in brain, heart, skeletal muscle, and spleen 1-3 DPI. Multiple genes were significantly upregulated following BCRV infection, including pattern recognition receptors (TLR7, TLR15, RIG-1), type I interferon (IFN-α), and type II interferon (IFN-γ). This is the first study to report avian immunological gene expression profiles following an arboviral infection.

  9. Structural basis for bifunctional peptide recognition at human δ-opioid receptor

    DOE PAGESBeta

    Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; et al

    2015-02-16

    Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. In summary, the observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

  10. Structural basis for bifunctional peptide recognition at human δ-opioid receptor

    SciTech Connect

    Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C. H.; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Gati, Cornelius; Yefanov, Oleksandr M.; White, Thomas A.; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N.; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W.; Roth, Bryan L.; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C.; Cherezov, Vadim

    2015-02-16

    Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. In summary, the observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

  11. Structural basis for bifunctional peptide recognition at human δ-Opioid receptor

    PubMed Central

    Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C.H.; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Gati, Cornelius; Yefanov, Oleksandr M.; White, Thomas A.; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N.; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W.; Roth, Bryan L.; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C.; Cherezov, Vadim

    2015-01-01

    Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt(1) and Tic(2). The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics. PMID:25686086

  12. Structural basis for bifunctional peptide recognition at human δ-opioid receptor.

    PubMed

    Fenalti, Gustavo; Zatsepin, Nadia A; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C H; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Gati, Cornelius; Yefanov, Oleksandr M; White, Thomas A; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W; Roth, Bryan L; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C; Cherezov, Vadim

    2015-03-01

    Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

  13. Structural basis for bifunctional peptide recognition at human δ-opioid receptor.

    PubMed

    Fenalti, Gustavo; Zatsepin, Nadia A; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C H; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Gati, Cornelius; Yefanov, Oleksandr M; White, Thomas A; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W; Roth, Bryan L; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C; Cherezov, Vadim

    2015-03-01

    Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics. PMID:25686086

  14. C-type lectin receptors Dectin-3 and Dectin-2 form a heterodimeric pattern-recognition receptor for host defense against fungal infection.

    PubMed

    Zhu, Le-Le; Zhao, Xue-Qiang; Jiang, Changying; You, Yun; Chen, Xiao-Ping; Jiang, Yuan-Ying; Jia, Xin-Ming; Lin, Xin

    2013-08-22

    C-type lectin receptors (CLRs) play critical roles as pattern-recognition receptors (PRRs) for sensing Candida albicans infection, which can be life-threatening for immunocompromised individuals. Here we have shown that Dectin-3 (also called CLECSF8, MCL, or Clec4d), a previously uncharacterized CLR, recognized α-mannans on the surfaces of C. albicans hyphae and induced NF-κB activation. Mice with either blockade or genetically deleted Dectin-3 were highly susceptible to C. albicans infection. Dectin-3 constantly formed heterodimers with Dectin-2, a well-characterized CLR, for recognizing C. albicans hyphae. Compared to their respective homodimers, Dectin-3 and Dectin-2 heterodimers bound α-mannans more effectively, leading to potent inflammatory responses against fungal infections. Together, our study demonstrates that Dectin-3 forms a heterodimeric PRR with Dectin-2 for sensing fungal infection and suggests that different CLRs may form different hetero- and homodimers, which provide different sensitivity and diversity for host cells to detect various microbial infections.

  15. Problem-Solving Test: Vitellogenin and Vitellogenin-Receptor Recognition: An Example of Protein Interaction

    ERIC Educational Resources Information Center

    Hernandez-Cortes, Patricia

    2012-01-01

    Vitellogenin (Vtg) is a lipid transfer protein that carries yolk to the ovary. The vitellogenin receptor (VtgR) mediates the uptake of Vtg into the oocyte of oviparous animals; its structure includes eight ligand-binding repeats (LBR). The binding site of VtgR and Vtg and the location of the interaction within the molecules are at these LBR.…

  16. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    SciTech Connect

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L.

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  17. Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.

    PubMed Central

    Boudin, H; Grauz-Guyon, A; Faure, M P; Forgez, P; Lhiaubet, A M; Dennis, M; Beaudet, A; Rostene, W; Pelaprat, D

    1995-01-01

    In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied

  18. Genetic polymorphisms in pattern recognition receptors and risk of periodontitis: Evidence based on 12,793 subjects.

    PubMed

    Han, Min-xuan; Ding, Cheng; Kyung, Hee-Moon

    2015-07-01

    Pattern recognition receptors (PRRs) constitute a pivotal arm of innate immunity. Many studies investigated the association between PRRs polymorphisms and periodontitis risk, which showed inconclusive results. The aim of the meta-analysis was to evaluate the precise association between five widely-evaluated polymorphisms (CD14 -260C/T (rs2569190), Toll-like receptor (TLR) 2 2408G/A (rs5743708), TLR4 896A/G (rs4986790), TLR4 1196C/T (rs4986791), mannose-binding lectin (MBL) codon 54 (rs1800450)) within the PRRs and susceptibility to either chronic (CP) or aggressive periodontitis (AgP). Overall, no significant association was found for the PRRs polymorphisms with either CP or AgP. In the subgroup analyses, TLR4 896G and 1196T alleles yielded a 32% (OR=1.32, 95% CI: 1.04-1.68) and a 37% increased CP risk (OR=1.37, 95% CI: 1.05-1.80) in Caucasians, respectively. Further stratified analyses revealed links between CD14, MBL2 polymorphisms and the severity of CP. This meta-analysis suggested that the periodontitis susceptibility was partly controlled by PRRs polymorphisms involved in the innate immunity.

  19. Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information.

    PubMed

    Paoletta, Silvia; Sabbadin, Davide; von Kügelgen, Ivar; Hinz, Sonja; Katritch, Vsevolod; Hoffmann, Kristina; Abdelrahman, Aliaa; Straßburger, Jens; Baqi, Younis; Zhao, Qiang; Stevens, Raymond C; Moro, Stefano; Müller, Christa E; Jacobson, Kenneth A

    2015-08-01

    The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

  20. The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots

    PubMed Central

    Holton, Nicholas; Nekrasov, Vladimir; Ronald, Pamela C.; Zipfel, Cyril

    2015-01-01

    During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots. PMID:25607985

  1. Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses

    DOE PAGESBeta

    Schwessinger, Benjamin; Bahar, Ofir; Thomas, Nicolas; Holton, Nicolas; Nekrasov, Vladimir; Ruan, Deling; Canlas, Patrick E.; Daudi, Arsalan; Petzold, Christopher J.; Singan, Vasanth R.; et al

    2015-03-30

    Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistancemore » to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.« less

  2. Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses

    PubMed Central

    Thomas, Nicolas; Holton, Nicolas; Nekrasov, Vladimir; Ruan, Deling; Canlas, Patrick E.; Daudi, Arsalan; Petzold, Christopher J.; Singan, Vasanth R.; Kuo, Rita; Chovatia, Mansi; Daum, Christopher; Heazlewood, Joshua L.; Zipfel, Cyril; Ronald, Pamela C.

    2015-01-01

    Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components. PMID:25821973

  3. Individual MHCI-Restricted T-Cell Receptors are Characterized by a Unique Peptide Recognition Signature.

    PubMed

    Wooldridge, Linda

    2013-01-01

    Effective immunity requires that a limited TCR repertoire is able to recognize a vast number of foreign peptide-MHCI (peptide-major histocompatibility complex class I) molecules. This challenge is overcome by the ability of individual TCRs to recognize large numbers of peptides. Recently, it was demonstrated that MHCI-restricted TCRs can recognize up to 10(6) peptides of a defined length. Astonishingly, this remarkable level of promiscuity does not extend to peptides of different lengths, a fundamental observation that has broad implications for CD8(+) T-cell immunity. In particular, the findings suggest that effective immunity can only be achieved by mobilization of "length-matched" CD8(+) T-cell clonotypes. Overall, recent findings suggest that every TCR is specific for a unique set of peptides, which can be described as a unique "peptide recognition signature" (PRS) and consists of three components: (1) peptide length preference, (2) number of peptides recognized; and, (3) sequence identity (e.g., self versus pathogen derived). In future, the ability to de-convolute peptide recognition signatures across the normal and pathogenic repertoire will be essential for understanding the system requirements for effective CD8(+) T-cell immunity and elucidating mechanisms which underlie CD8(+) T-cell mediated disease.

  4. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  5. Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor

    PubMed Central

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A. J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-01-01

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein–coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state–like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state. PMID:25745166

  6. Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

    PubMed Central

    Rios, Pablo; Carter, Tom S.; Crump, Matthew P.; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T.; Boons, Geert‐Jan

    2016-01-01

    Abstract The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K a≈20 000 m −1, whereas the other one binds an O‐GlcNAcylated peptide with K a≈70 000 m −1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts. PMID:26822115

  7. Murine pattern recognition receptor dectin-1 is essential in the development of experimental autoimmune uveoretinitis.

    PubMed

    Stoppelkamp, Sandra; Reid, Delyth M; Yeoh, Joyce; Taylor, Julie; McKenzie, Emma J; Brown, Gordon D; Gordon, Siamon; Forrester, John V; Wong, Simon Y C

    2015-10-01

    Mycobacteria in complete Freund's adjuvant (CFA) are an essential component of immunization protocols in a number of autoimmune disease animal models including experimental autoimmune encephalomyelitis and uveoretinitis (EAE and EAU, respectively). We determined the role in EAU of two C-type lectin receptors on myeloid cells that recognize and respond to mycobacteria. Using receptor-specific antibodies and knockout mice, we demonstrated for the first time that the macrophage mannose receptor delays disease development but does not affect severity. In contrast, dectin-1 is critically involved in the development of CFA-mediated EAU. Disease severity is reduced in dectin-1 knockout mice and antibody blockade of dectin-1 during the induction, but not the effector phase, prevents EAU development. Significantly, similar blockade of dectin-1 in vivo has no effect in non-CFA-mediated, spontaneously induced or adoptive transfer models of EAU. Thus dectin-1 plays a critical role in the ability of complete Freund's adjuvant to induce EAU in mice.

  8. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2

    SciTech Connect

    Armstrong, Anthony A.; Mohideen, Firaz; Lima, Christopher D.

    2013-04-08

    Ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers such as SUMO (also known as Smt3 in Saccharomyces cerevisiae) mediate signal transduction through post-translational modification of substrate proteins in pathways that control differentiation, apoptosis and the cell cycle, and responses to stress such as the DNA damage response. In yeast, the proliferating cell nuclear antigen PCNA (also known as Pol30) is modified by ubiquitin in response to DNA damage and by SUMO during S phase. Whereas Ub-PCNA can signal for recruitment of translesion DNA polymerases, SUMO-PCNA signals for recruitment of the anti-recombinogenic DNA helicase Srs2. It remains unclear how receptors such as Srs2 specifically recognize substrates after conjugation to Ub and Ubls. Here we show, through structural, biochemical and functional studies, that the Srs2 carboxy-terminal domain harbors tandem receptor motifs that interact independently with PCNA and SUMO and that both motifs are required to recognize SUMO-PCNA specifically. The mechanism presented is pertinent to understanding how other receptors specifically recognize Ub- and Ubl-modified substrates to facilitate signal transduction.

  9. Effect of aging on microRNAs and regulation of pathogen recognition receptors.

    PubMed

    Olivieri, Fabiola; Procopio, Antonio Domenico; Montgomery, Ruth R

    2014-08-01

    Immunosenescence is the multifactorial age-associated immune deteriorization that leads to increased susceptibility to infections and decreased responses to vaccines. Recent studies have shown a fundamental role for microRNAs (miRNAs) in regulating immune responses, and nearly all the miRNAs involved in immune regulation show modulation during aging. Aging-associated miRNAs are largely negative regulators of the immune innate response and target central nodes of aging-associated networks, in particular, NF-κB, the downstream effector of TLR signals that leads to induction of proinflammatory responses. Multiple miRNAs have been reported to share similar regulatory activity. Here we review miRNA regulation of human innate immune recognition in aging, including both activation and resolution of inflammation, critical issues in detection, and areas of active investigation into our understanding of immunosenescence.

  10. Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity

    PubMed Central

    Lee, Peter S.; Yoshida, Reiko; Ekiert, Damian C.; Sakai, Naoki; Suzuki, Yasuhiko; Takada, Ayato; Wilson, Ian A.

    2012-01-01

    Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes. PMID:23027945

  11. Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response

    PubMed Central

    Paramo, Teresa; Tomasio, Susana M.; Irvine, Kate L.; Bryant, Clare E.; Bond, Peter J.

    2015-01-01

    Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics. PMID:26647780

  12. Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

    PubMed

    Paramo, Teresa; Tomasio, Susana M; Irvine, Kate L; Bryant, Clare E; Bond, Peter J

    2015-01-01

    Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the "membrane-like" nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics. PMID:26647780

  13. Cellular Recognition and Trafficking of Amorphous Silica Nanoparticles by Macrophage Scavenger Receptor A

    SciTech Connect

    Orr, Galya; Chrisler, William B.; Cassens, Kaylyn J.; Tan, Ruimin; Tarasevich, Barbara J.; Markillie, Lye Meng; Zangar, Richard C.; Thrall, Brian D.

    2011-09-01

    The internalization of engineered nanoparticles (ENPs) into cells is known to involve active transport mechanisms, yet the precise biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs (92 nm) by macrophage cells is strongly inhibited by silencing expression of scavenger receptor A (SR-A). In addition, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal fluorescent microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize intracellularly with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica NP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting independent trafficking or internalization pathways are involved. SR-A silencing also caused decreased cellular secretion of pro-inflammatory cytokines in response to silica ENPs. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biodistribution of, and cellular response to ENPs.

  14. Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle.

    PubMed

    Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

    2014-06-21

    Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of 'exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of 'endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of 'endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.

  15. Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

    PubMed

    Paramo, Teresa; Tomasio, Susana M; Irvine, Kate L; Bryant, Clare E; Bond, Peter J

    2015-12-09

    Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the "membrane-like" nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.

  16. Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses.

    PubMed

    Abraham, Jonathan; Corbett, Kevin D; Farzan, Michael; Choe, Hyeryun; Harrison, Stephen C

    2010-04-01

    New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

  17. Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

    SciTech Connect

    Abraham, Jonathan; Corbett, Kevin D.; Farzan, Michael; Choe, Hyeryun; Harrison, Stephen C.

    2010-08-18

    New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

  18. Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

    PubMed

    Burden-Gulley, Susan M; Qutaish, Mohammed Q; Sullivant, Kristin E; Tan, Mingqian; Craig, Sonya E L; Basilion, James P; Lu, Zheng-Rong; Wilson, David L; Brady-Kalnay, Susann M

    2013-04-01

    Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.

  19. Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

    PubMed

    Burden-Gulley, Susan M; Qutaish, Mohammed Q; Sullivant, Kristin E; Tan, Mingqian; Craig, Sonya E L; Basilion, James P; Lu, Zheng-Rong; Wilson, David L; Brady-Kalnay, Susann M

    2013-04-01

    Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells. PMID:22987116

  20. Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals.

    PubMed

    Deblonde, Gauthier J-P; Sturzbecher-Hoehne, Manuel; Mason, Anne B; Abergel, Rebecca J

    2013-06-01

    Following an internal contamination event, the transport of actinide (An) and lanthanide (Ln) metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe(3+), Ga(3+), La(3+), Nd(3+), Gd(3+), Yb(3+), Lu(3+), (232)Th(4+), (238)UO2(2+), and (242)Pu(4+). Important features of this method are (i) its ability to distinguish both 1 : 1 and 1 : 2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 μM and Kd2 = 1.8 μM) binding to the TfR. Other toxic metal ions such as Th(IV) and U(VI), when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe(3+) > Th(4+) ~ UO2(2+) ~ Cm(3+) > Ln(3+) ~ Ga(3+) > Yb(3+) ~ Pu(4+). This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor-mediated endocytosis. PMID:23446908

  1. Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals.

    PubMed

    Deblonde, Gauthier J-P; Sturzbecher-Hoehne, Manuel; Mason, Anne B; Abergel, Rebecca J

    2013-06-01

    Following an internal contamination event, the transport of actinide (An) and lanthanide (Ln) metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe(3+), Ga(3+), La(3+), Nd(3+), Gd(3+), Yb(3+), Lu(3+), (232)Th(4+), (238)UO2(2+), and (242)Pu(4+). Important features of this method are (i) its ability to distinguish both 1 : 1 and 1 : 2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 μM and Kd2 = 1.8 μM) binding to the TfR. Other toxic metal ions such as Th(IV) and U(VI), when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe(3+) > Th(4+) ~ UO2(2+) ~ Cm(3+) > Ln(3+) ~ Ga(3+) > Yb(3+) ~ Pu(4+). This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor-mediated endocytosis.

  2. Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals

    PubMed Central

    Deblonde, Gauthier J.-P.; Sturzbecher-Hoehne, Manuel; Mason, Anne B.; Abergel, Rebecca J.

    2013-01-01

    Following an internal contamination event, the transport of actinide and lanthanide metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe3+, Ga3+, La3+, Nd3+, Gd3+, Yb3+, Lu3+, 232Th4+, 238UO22+, and 242Pu4+. Important features of this method are (i) its ability to distinguish both 1:1 and 1:2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 µM and Kd2 = 1.8 µM) binding to the TfR. Other toxic metal ions such as ThIV and UVI, when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe3+ >> Th4+ □ UO22+ □ Cm3+ > Ln3+ □ Ga3+ >>> Yb3+ □ Pu4+. This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor mediated endocytosis. PMID:23446908

  3. Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3*

    PubMed Central

    Mirza, Shamaruh; Chen, Jinglong; Wen, Bin; Ewens, Cameron L.; Dai, Jin; Murphy, James M.; Young, Ian G.

    2010-01-01

    The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (βIL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length (“SP1” isoform) and a novel isoform (denoted “SP2”) lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the βIL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of βIL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu23, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the βIL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of βIL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes. PMID:20472554

  4. Teratogenesis of polychlorocycloalkane insecticides in chicken embryos resulting from their interactions at the convulsant recognition sites of the GABA (pro)receptor complex

    SciTech Connect

    Seifert, J. )

    1989-05-01

    There is an increasing concern for the effects of toxicants expressed during embryonic development. This paper considers the teratogenicity of polychlorocycloalkane insecticides. They are neurotoxicants which act at the convulsant recognition site(s) of the GABA receptor-chloride ionophore and antagonize GABA-mediated inhibitory neurotransmission. This study uses the chicken embryo as a model organism. It focuses on malformations related to interactions of polychlorocycloalkane insecticides with the convulsant recognition sites in the brain. Information provided by this study is important for investigations on the teratogenicity of insecticidal GABA antagonists in mammals.

  5. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-05-15

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  6. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed Central

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-01-01

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  7. Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle

    NASA Astrophysics Data System (ADS)

    Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

    2014-05-01

    Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied

  8. Molecular Recognition and Scavenging of Arsenate from Aqueous Solution Using Dimetallic Receptors

    PubMed Central

    Moffat, Chris D; Weiss, Dominik J; Shivalingam, Arun; White, Andrew J P; Salaün, Pascal; Vilar, Ramon

    2014-01-01

    A series of copper(II), nickel(II) and zinc(II) dimetallic complexes were prepared and their affinities towards arsenate investigated. Indicator displacement assays (IDAs) were carried out to establish the complexes with best affinities towards arsenate. A di-zinc complex (3) was selected and its arsenate-binding abilities investigated by isothermal titration calorimetry (ITC). The X-ray crystal structure of this metallo-receptor bound to arsenate is also reported, which allowed us to establish the binding mode between 3 and this oxyanion. Immobilising 3 onto HypoGel resin yielded a novel adsorbent (Zn–HypoGel) with high affinity for arsenate. Adsorption of arsenate from competitive solutions and natural groundwater was greater than that of the commercially used iron oxide Bayoxide E33. Zn–HypoGel could be efficiently and simply regenerated by washing with sodium acetate solution. PMID:25338508

  9. Function of Nod-like Receptors in Microbial Recognition and Host Defense

    PubMed Central

    Franchi, Luigi; Warner, Neil; Viani, Kyle; Nuñez, Gabriel

    2009-01-01

    Summary Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a specialized group of intracellular proteins that play a critical role in the regulation of the host innate immune response. NLRs act as scaffolding proteins that assemble signaling platforms that trigger nuclear factor-κB and mitogen-activated protein kinase signaling pathways and control the activation of inflammatory caspases. Importantly, mutations in several members of the NLR family have been linked to a variety of inflammatory diseases consistent with these molecules playing an important role in host-pathogen interactions and the inflammatory response. In this review, we focus on the role of Nod1 and Nod2 in host defense and in particular discuss recent finding regarding the role of Nlrc4, Nlpr1, and Nlrp3 inflammasomes in caspase-1 activation and subsequent release of proinflammatory cytokines such as interleukin-1β. PMID:19120480

  10. Computational evaluation of unsaturated carbonitriles as neutral receptor model for beryllium(II) recognition.

    PubMed

    Rosli, Ahmad Nazmi; Ahmad, Mohd Rais; Alias, Yatimah; Zain, Sharifuddin Md; Lee, Vannajan Sanghiran; Woi, Pei Meng

    2014-12-01

    Design of neutral receptor molecules (ionophores) for beryllium(II) using unsaturated carbonitrile models has been carried out via density functional theory, G3, and G4 calculations. The first part of this work focuses on gas phase binding energies between beryllium(II) and 2-cyano butadiene (2-CN BD), 3-cyano propene (3-CN P), and simpler models with two separate fragments; acrylonitrile and ethylene. Interactions between beryllium(II) and cyano nitrogen and terminal olefin in the models have been examined in terms of geometrical changes, distribution of charge over the entire π-system, and rehybridization of vinyl carbon orbitals. NMR shieldings and vibrational frequencies probed charge centers and strength of interactions. The six-membered cyclic complexes have planar structures with the rehybridized carbon slightly out of plane (16° in 2-CN BD). G3 results show that in 2-CN BD complex participation of vinyl carbon further stabilizes the cyclic adduct by 16.3 kcal mol(-1), whereas, in simpler models, interaction between beryllium(II) and acetonitrile is favorable by 46.4 kcal mol(-1) compared with that of ethylene. The terminal vinyl carbon in 2-CN BD rehybridizes to sp (3) with an increase of 7 % of s character to allow interaction with beryllium(II). G4 calculations show that the Be(II) and 2-CN BD complex is more strongly bound than those with Mg(II) and Ca(II) by 98.5 and 139.2 kcal mol(-1) (-1), respectively. QST2 method shows that the cyclic and acyclic forms of Be(II)-2-CN BD complexes are separated by 12.3 kcal mol(-1) barrier height. Overlap population analysis reveals that Ca(II) can be discriminated based on its tendency to form ionic interaction with the receptor models.

  11. Urea-Functionalized M4L6 Cage Receptors: Self-Assembly, Dynamics, and Anion Recognition in Aqueous Solutions

    SciTech Connect

    Custelcean, Radu; Bonnesen, Peter V; Duncan, Nathan C; Van Berkel, Gary J; Hay, Benjamin

    2012-01-01

    We present an extensive study of a novel class of de novo designed tetrahedral M{sub 4}L{sub 6} (M = Ni, Zn) cage receptors, wherein internal decoration of the cage cavities with urea anion-binding groups, via functionalization of the organic components L, led to selective encapsulation of tetrahedral oxoanions EO{sub 4}{sup -} (E = S, Se, Cr, Mo, W, n = 2; E = P, n = 3) from aqueous solutions, based on shape, size, and charge recognition. External functionalization with tBu groups led to enhanced solubility of the cages in aqueous methanol solutions, thereby allowing for their thorough characterization by multinuclear ({sup 1}H, {sup 13}C, {sup 77}Se) and diffusion NMR spectroscopies. Additional experimental characterization by electrospray ionization mass spectrometry, UV-vis spectroscopy, and single-crystal X-ray diffraction, as well as theoretical calculations, led to a detailed understanding of the cage structures, self-assembly, and anion encapsulation. We found that the cage self-assembly is templated by EO{sub 4}{sup -} oxoanions (n {ge} 2), and upon removal of the templating anion the tetrahedral M{sub 4}L{sub 6} cages rearrange into different coordination assemblies. The exchange selectivity among EO{sub 4}{sup -} oxoanions has been investigated with {sup 77}Se NMR spectroscopy using {sup 77}SeO{sub 4}{sup 2-} as an anionic probe, which found the following selectivity trend: PO{sub 4}{sup 3-} CrO{sub 4}{sup 2-} > SO{sub 4}{sup 2-} > SeO{sub 4}{sup 2-} > MoO{sub 4}{sup 2-} > WO{sub 4}{sup 2-}. In addition to the complementarity and flexibility of the cage receptor, a combination of factors have been found to contribute to the observed anion selectivity, including the anions charge, size, hydration, basicity, and hydrogen-bond acceptor abilities.

  12. Aberrant expression of regulatory cytokine IL-35 and pattern recognition receptor NOD2 in patients with allergic asthma.

    PubMed

    Wong, Chun Kwok; Leung, Ting Fan; Chu, Ida Miu Ting; Dong, Jie; Lam, Yvonne Yi On; Lam, Christopher Wai Kei

    2015-02-01

    We investigated the plasma concentration of the novel regulatory cytokine IL-35 and intracytosolic pattern recognition receptors nucleotide-binding oligomerization domain (NOD)-like receptors in granulocytes and explored their potential implication in disease severity monitoring of allergic asthma. The expression of circulating IL-35 and other pro-inflammatory mediators in asthmatic patients or control subjects were evaluated using enzyme-linked immunosorbent assay (ELISA). The intracellular expressions of NOD1 and NOD2 in CCR3+ granulocytes were assessed using flow cytometry. Plasma concentrations of IL-35, IL-17A, basophil activation marker basogranulin, and eosinophilic airway inflammation biomarker periostin were significantly elevated in allergic asthmatic patients compared to non-atopic control subjects (all probability (p) <0.05). Both granulocyte markers exhibited significant and positive correlation with plasma IL-35 concentration in asthmatic patients (all p < 0.05). Significant positive correlation was also identified between plasma concentrations of IL-35 and periostin with disease severity score in asthmatic patients (both p < 0.05). The basophil activation allergenicity test was positive in allergic asthmatic patients but not in control subjects. Despite significantly elevated eosinophil count in allergic asthmatic patients, downregulation of NOD2 in CCR3+ granulocytes was observed in these patients (both p < 0.05). A negative correlation between plasma concentrations of tumor necrosis factor family member LIGHT and soluble herpesvirus entry mediator was observed in patients with elevated plasma concentration of IL-35 (p < 0.05). Aberrant expression of NOD2 in granulocytes may be contributed to the impaired innate immunity predisposing allergic asthma. IL-35 may serve as a potential surrogate biomarker for disease severity of allergic asthma.

  13. The incidence and significance of pattern-recognition receptors in chronic viral hepatitis types B and C in man.

    PubMed

    Mozer-Lisewska, Iwona; Sikora, Jan; Kowala-Piaskowska, Arleta; Kaczmarek, Mariusz; Dworacki, Grzegorz; Zeromski, Jan

    2010-08-01

    Chronic viral hepatitis B and C are among the most common and devastating liver diseases worldwide. Immune response plays a crucial role in the course of both diseases. In spite of the importance of the adaptive arm of the immune response, there is a growing role of innate immunity, the earliest confronted with viral attack. Pattern-recognition receptors (PRRs) and, in particular, Toll-like receptors (TLRs) are molecules which are able not only to recognize foreign invaders, but also quickly mount an antiviral defense. Activation of PRRs has been demonstrated in both hepatitis types, i.e. in situ in the liver and on while blood cells. Both viruses, HCV and HBV, are able to subvert the PRR-mediated antiviral response by means of various proteins and enzymes. HCV acts via the non-structural proteins NS2 and NS3/4A, while HBV HBeAg is inversely correlated with TLR activity. Viral counterattack is particularly directed toward dendritic cells, those creating the link with the adaptive immune response. Apart from TLRs, other PRRs such as RIG-1 and MDA-5 are also able to recognize viral infection and participate in the activation of type I interferon synthesis. TLRs manifest gene polymorphism, which was shown to affect several consequences associated with chronic viral hepatitis such as liver cirrhosis and the outcome of liver allotransplantation. There have been numerous attempts to take advantage of the existence and activity of PRRs for the patients' benefit. Several authors examined the role of TLR synthetic agonists as inducers of TLR activation. In hepatitis C the most promising agonists appear to be TLR3, 7, and 9 for potential antiviral therapy. PRRs may also act as potent adjuvants in HBV vaccines. Their baseline mRNA levels may have predictive value in the course of antiviral therapy.

  14. Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses

    SciTech Connect

    Schwessinger, Benjamin; Bahar, Ofir; Thomas, Nicolas; Holton, Nicolas; Nekrasov, Vladimir; Ruan, Deling; Canlas, Patrick E.; Daudi, Arsalan; Petzold, Christopher J.; Singan, Vasanth R.; Kuo, Rita; Chovatia, Mansi; Daum, Christopher; Heazlewood, Joshua L.; Zipfel, Cyril; Ronald, Pamela C.

    2015-03-30

    Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.

  15. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    PubMed Central

    López-Sagaseta, Jacinto; Sibener, Leah V; Kung, Jennifer E; Gumperz, Jenny; Adams, Erin J

    2012-01-01

    Invariant Natural Killer T (iNKT) cells use highly restricted αβ T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted α1 helix resulting in an open A' pocket. Binding of the iNKT TCR requires a 7-Å displacement of the LPC headgroup but stabilizes the CD1d–LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d–LPC is anchored by the conserved positioning of the CDR3α loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3β and Jβ segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells. PMID:22395072

  16. The soluble pattern recognition receptor PTX3 links humoral innate and adaptive immune responses by helping marginal zone B cells.

    PubMed

    Chorny, Alejo; Casas-Recasens, Sandra; Sintes, Jordi; Shan, Meimei; Polentarutti, Nadia; García-Escudero, Ramón; Walland, A Cooper; Yeiser, John R; Cassis, Linda; Carrillo, Jorge; Puga, Irene; Cunha, Cristina; Bastos, Hélder; Rodrigues, Fernando; Lacerda, João F; Morais, António; Dieguez-Gonzalez, Rebeca; Heeger, Peter S; Salvatori, Giovanni; Carvalho, Agostinho; Garcia-Sastre, Adolfo; Blander, J Magarian; Mantovani, Alberto; Garlanda, Cecilia; Cerutti, Andrea

    2016-09-19

    Pentraxin 3 (PTX3) is a fluid-phase pattern recognition receptor of the humoral innate immune system with ancestral antibody-like properties but unknown antibody-inducing function. In this study, we found binding of PTX3 to splenic marginal zone (MZ) B cells, an innate-like subset of antibody-producing lymphocytes strategically positioned at the interface between the circulation and the adaptive immune system. PTX3 was released by a subset of neutrophils that surrounded the splenic MZ and expressed an immune activation-related gene signature distinct from that of circulating neutrophils. Binding of PTX3 promoted homeostatic production of IgM and class-switched IgG antibodies to microbial capsular polysaccharides, which decreased in PTX3-deficient mice and humans. In addition, PTX3 increased IgM and IgG production after infection with blood-borne encapsulated bacteria or immunization with bacterial carbohydrates. This immunogenic effect stemmed from the activation of MZ B cells through a neutrophil-regulated pathway that elicited class switching and plasmablast expansion via a combination of T cell-independent and T cell-dependent signals. Thus, PTX3 may bridge the humoral arms of the innate and adaptive immune systems by serving as an endogenous adjuvant for MZ B cells. This property could be harnessed to develop more effective vaccines against encapsulated pathogens. PMID:27621420

  17. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    SciTech Connect

    López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.; Gumperz, Jenny; Adams, Erin J.

    2014-10-02

    Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

  18. Treatment of chronic hepatitis B with pattern recognition receptor agonists: Current status and potential for a cure

    PubMed Central

    Chang, Jinhong; Guo, Ju-Tao

    2015-01-01

    Hepatitis B virus (HBV) has been considered to be a “stealth virus” that induces negligible innate immune responses during the early phase of infection. However, recent studies with newly developed experimental systems have revealed that virus infection can be recognized by pattern recognition receptors (PRR), eliciting a cytokine response that controls the replication of the virus. The molecular mechanisms by which interferons and other inflammatory cytokines suppress HBV replication and modulate HBV cccDNA metabolism and function are just beginning to be revealed. In agreement with the notion that the developmental and functional status of intrahepatic innate immunity determines the activation and maturation of the HBV-specific adaptive immune response and thus the outcome of HBV infection, pharmacological activation of intrahepatic innate immune responses with TLR7/8/9 or STING agonists efficiently controls HBV infection in preclinical studies and thus holds great promise for the cure of chronic hepatitis B. This article forms part of a symposium in Antiviral Research on “An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B.” PMID:26205674

  19. Buggy Creek Virus (Togaviridae: Alphavirus) Upregulates Expression of Pattern Recognition Receptors and Interferons in House Sparrows (Passer domesticus)

    PubMed Central

    Barak, Virginia A.; Rainwater, Ellecia L.; Altrichter, Ashley M.

    2014-01-01

    Abstract Birds serve as reservoirs for at least 10 arthropod-borne viruses, yet specific immune responses of birds to arboviral infections are relatively unknown. Here, adult House Sparrows were inoculated with an arboviral alphavirus, Buggy Creek virus (BCRV), or saline, and euthanized between 1 and 3 days postinoculation. Virological dynamics and gene expression dynamics were investigated. Birds did not develop viremia postinoculation, but cytopathic virus was found in the skeletal muscle and spleen of birds 1 and 3 days postinoculation (DPI). Viral RNA was detected in the blood of BCRV-infected birds 1 and 2 DPI, in oral swabs 1–3 DPI, and in brain, heart, skeletal muscle, and spleen 1–3 DPI. Multiple genes were significantly upregulated following BCRV infection, including pattern recognition receptors (TLR7, TLR15, RIG-1), type I interferon (IFN-α), and type II interferon (IFN-γ). This is the first study to report avian immunological gene expression profiles following an arboviral infection. PMID:24866749

  20. Single cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents

    PubMed Central

    Smolen, Kinga K; Cai, Bing; Fortuno, Edgardo S; Gelinas, Laura; Larsen, Martin; Speert, David P; Chamekh, Mustapha; Kollmann, Tobias R

    2014-01-01

    Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with increased risk for infection. We had previously shown that whole blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors (PRR) as compared to whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we now set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, significant reduction of the intracellular cytokine production on the single cell level was only detected in South African childrens’ monocytes, cDC, and pDC. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children as compared to children from other continents. Together our data identify differences in cell composition as well as profoundly lower functional responses of innate cells in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored. PMID:25135829

  1. Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.

    PubMed

    Fan, Chunxin; Zhang, Shicui; Li, Lei; Chao, Yeqing

    2008-07-01

    Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus (Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen beta and gamma chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response. PMID:18533266

  2. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA.

    PubMed

    Kowalinski, Eva; Lunardi, Thomas; McCarthy, Andrew A; Louber, Jade; Brunel, Joanna; Grigorov, Boyan; Gerlier, Denis; Cusack, Stephen

    2011-10-14

    RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.

  3. Inactivated vaccine with adjuvants consisting of pattern recognition receptor agonists confers protection against avian influenza viruses in chickens.

    PubMed

    Tang, Yinghua; Lu, Jihu; Wu, Peipei; Liu, Zhenxing; Tian, Zhen; Zha, Guofei; Chen, Hui; Wang, Qiaochu; Wang, Qiaoxiu; Hou, Fengxiang; Kang, Sang-Moo; Hou, Jibo

    2014-08-01

    Use of adjuvant containing pathogen pattern recognition receptor agonists is one of the effective strategies to enhance the efficacy of licensed vaccines. In this study, we investigated the efficacy of avian influenza vaccines containing an adjuvant (CVCVA5) which was composed of polyriboinosinic polyribocytidylic, resiquimod, imiquimod, muramyl dipeptide and levomisole. Avian influenza vaccines adjuvanted with CVCVA5 were found to induce significantly higher titers of hemagglutiniton inhibition antibodies (P≤0.01) than those of commercial vaccines at 2-, 3- and 4-week post vaccination in both specific pathogen free (SPF) chickens and field application. Furthermore, virus shedding was reduced in SPF chickens immunized with H9-CVCVA5 vaccine after H9 subtype heterologous virus challenge. The ratios of both CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes were slowly elevated in chickens immunized with H9-CVCVA5 vaccine. Lymphocytes adoptive transfer study indicates that CD8(+) T lymphocyte subpopulation might have contributed to improved protection against heterologous virus challenge. Results of this study suggest that the adjuvant CVCVA5 was capable of enhancing the potency of existing avian influenza vaccines by increasing humoral and cellular immune response.

  4. Treatment of chronic hepatitis B with pattern recognition receptor agonists: Current status and potential for a cure.

    PubMed

    Chang, Jinhong; Guo, Ju-Tao

    2015-09-01

    Hepatitis B virus (HBV) has been considered to be a "stealth virus" that induces negligible innate immune responses during the early phase of infection. However, recent studies with newly developed experimental systems have revealed that virus infection can be recognized by pattern recognition receptors (PRR), eliciting a cytokine response that controls the replication of the virus. The molecular mechanisms by which interferons and other inflammatory cytokines suppress HBV replication and modulate HBV cccDNA metabolism and function are just beginning to be revealed. In agreement with the notion that the developmental and functional status of intrahepatic innate immunity determines the activation and maturation of the HBV-specific adaptive immune response and thus the outcome of HBV infection, pharmacological activation of intrahepatic innate immune responses with TLR7/8/9 or STING agonists efficiently controls HBV infection in preclinical studies and thus holds great promise for the cure of chronic hepatitis B. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."

  5. Inactivated vaccine with adjuvants consisting of pattern recognition receptor agonists confers protection against avian influenza viruses in chickens

    PubMed Central

    Tang, Yinghua; Lu, Jihu; Wu, Peipei; Liu, Zhenxing; Tian, Zhen; Zha, Guofei; Chen, Hui; Wang, Qiaochu; Wang, Qiaoxiu; Hou, Fengxiang; Kang, Sang-Moo; Hou, Jibo

    2014-01-01

    Use of adjuvant containing pathogen pattern recognition receptor agonists is one of the effective strategies to enhance the efficacy of licensed vaccines. In this study, we investigated the efficacy of avian influenza vaccines containing an adjuvant (CVCVA5) which was composed of polyriboinosinic polyribocytidylic, resiquimod, imiquimod, muramyl dipeptide and levomisole. Avian influenza vaccines adjuvanted with CVCVA5 were found to induce significantly higher titers of hemagglutiniton inhibition antibodies (P ≤ 0.01) than those of commercial vaccines at 2-, 3- and 4-week post vaccination in both specific pathogen free (SPF) chickens and field application. Furthermore, virus shedding was reduced in SPF chickens immunized with H9-CVCVA5 vaccine after H9 subtype heterologous virus challenge. The ratios of both CD3+CD4+ and CD3+CD8+ lymphocytes were slowly elevated in chickens immunized with H9-CVCVA5 vaccine. Lymphocytes adoptive transfer study indicates that CD8+ T lymphocyte subpopulation might have contributed to improved protection against heterologous virus challenge. Results of this study suggest that the adjuvant CVCVA5 was capable of enhancing the potency of existing avian influenza vaccines by increasing humoral and cellular immune response. PMID:24894132

  6. Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle*

    PubMed Central

    Feinberg, Hadar; Rambaruth, Neela D. S.; Jégouzo, Sabine A. F.; Jacobsen, Kristian M.; Djurhuus, Rasmus; Poulsen, Thomas B.; Weis, William I.; Taylor, Maureen E.; Drickamer, Kurt

    2016-01-01

    The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages. PMID:27542410

  7. Single-cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents.

    PubMed

    Smolen, Kinga K; Cai, Bing; Gelinas, Laura; Fortuno, Edgardo S; Larsen, Martin; Speert, David P; Chamekh, Mustapha; Cooper, Philip J; Esser, Monika; Marchant, Arnaud; Kollmann, Tobias R

    2014-09-15

    Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with an increased risk for infection. We showed previously that whole-blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors compared with whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, a significant reduction in the intracellular cytokine production on the single-cell level was only detected in South African children's monocytes, conventional dendritic cells, and plasmacytoid dendritic cells. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children compared with children from the other continents. Together, our data identify differences in cell composition, as well as profoundly lower functional responses of innate cells, in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored.

  8. The Inorganic Perspective of VEGF: Interactions of Cu(2+) with Peptides Encompassing a Recognition Domain of the VEGF Receptor.

    PubMed

    Grasso, Giulia; Santoro, Anna Maria; Magrì, Antonio; La Mendola, Diego; Tomasello, Marianna Flora; Zimbone, Stefania; Rizzarelli, Enrico

    2016-06-01

    The vascular endothelial growth factor A (VEGF-A) is a potent angiogenic factor, its activity may be influenced by the presence of copper(II) ions. To mimic the interaction between copper(II) and VEGF (Vascular Endotelial Growth Factor), the N- and C-terminally blocked peptide fragments VEGF73-101 and VEGF84-101, owing to VEGF165 protein, have been synthesized. These protein domains represent a specific recognition site with the VEGF receptor (VEGFR). Copper(II) complexes with VEGF73-101 and VEGF84-101 were investigated by means of potentiometry and UV-Vis, ESI-MS, CD, EPR spectroscopic methods. Both peptides have three histidine residues and display a binding high affinity for copper(II) ions. The proliferative activity of the peptides in the absence and presence of copper(II) ions as well as of VEGF-165 protein was also tested on HUVEC cells (Human Umbilical Vein Endothelial Cells). The VEGF73-101 showed a dose-dependent anti-proliferative activity, while the shorter peptide VEGF84-101 did not affect HUVEC proliferation, both in the presence and in the absence of VEGF. PMID:27015654

  9. Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses

    PubMed Central

    Wang, Qiong; Nagarkar, Deepti R.; Bowman, Emily R.; Schneider, Dina; Gosangi, Babina; Lei, Jing; Zhao, Ying; McHenry, Christina L.; Burgens, Richai V.; Miller, David J.; Sajjan, Umadevi; Hershenson, Marc B.

    2010-01-01

    Rhinovirus (RV), a single-stranded RNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral double-stranded RNA produced during replication may be recognized by the host pattern recognition receptors Toll-like receptor (TLR)-3, retinoic acid inducible gene (RIG)-I and melanoma-differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV double-stranded (ds)-RNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and interferon (IFN) and IFN-stimulated gene expression determined by quantitative PCR. The separate requirements of RIG-I, MDA5 and IFN response factor (IRF)-3 were determined using their respective siRNAs. The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule TRIF. Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β̤, IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5, IP-10/CXCL10, IL-8/CXCL8 and GM-CSF. siRNA against IRF3, MDA5 and TRIF, but not RIG-I, decreased RV1B-induced expression of IFN-β̤ IFN-λ̣1, IFN-λ2/3, IRF7, RIG-I, MDA5 and IP-10/CXCL10, but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA, and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3. PMID:19890046

  10. IL-1R signaling in dendritic cells replaces pattern-recognition receptors in promoting CD8⁺ T cell responses to influenza A virus.

    PubMed

    Pang, Iris K; Ichinohe, Takeshi; Iwasaki, Akiko

    2013-03-01

    Immune responses to vaccines require direct recognition of pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs) on dendritic cells (DCs). Unlike vaccination, infection by a live pathogen often impairs DC function and inflicts additional damage on the host. Here we found that after infection with live influenza A virus, signaling through the interleukin 1 receptor (IL-1R) was required for productive priming of CD8(+) T cells, but signaling through the PRRs TLR7 and RIG-I was not. DCs activated by IL-1 in trans were both required and sufficient for the generation of virus-specific CD8(+) T cell immunity. Our data demonstrate a critical role for a bystander cytokine in the priming of CD8(+) T cells during infection with a live virus.

  11. The melanin-concentrating hormone1 receptor antagonists, SNAP-7941 and GW3430, enhance social recognition and dialysate levels of acetylcholine in the frontal cortex of rats.

    PubMed

    Millan, Mark J; Gobert, Alain; Panayi, Fany; Rivet, Jean-Michel; Dekeyne, Anne; Brocco, Mauricette; Ortuno, Jean-Claude; Di Cara, Benjamin

    2008-12-01

    Melanin-concentrating hormone (MCH)1 receptors are widely expressed in limbic structures and cortex. Their inactivation is associated with anxiolytic and antidepressive properties but little information is available concerning cognition. This issue was addressed using the selective antagonists, SNAP-7941 and GW3430, in a social recognition paradigm in rats. The muscarinic blocker, scopolamine (1.25 mg/kg s.c.), reduced social recognition, an action dose-dependently blocked by SNAP-7941 and GW3430 (0.63-10.0 and 20.0-80.0 mg/kg i.p., respectively) which did not themselves display amnesic properties. Further, in a protocol where a spontaneous deficit was induced by a prolonged inter-session delay, SNAP-7941 and GW3430 dose-dependently enhanced social recognition. In dialysis studies, SNAP-7941 (0.63-40.0 mg/kg i.p.) and GW3430 (10.0-40.0 mg/kg i.p.) elevated extracellular levels of acetylcholine (ACh) in the frontal cortex (FCX) of freely moving rats. The SNAP-7941 effect was specific, as it did not increase levels of ACh in ventral and dorsal hippocampus: moreover, it did not modify levels of noradrenaline, dopamine, serotonin and glutamate in FCX. Active doses of SNAP-7941 and GW3430 corresponded to doses (2.5-40.0 and 10.0-80.0 mg/kg i.p., respectively) exerting anxiolytic properties in Vogel conflict and ultrasonic vocalization tests, and antidepressant actions in forced swim, isolation-induced aggression and marble-burying procedures. In contrast to SNAP-7941 and GW3430, the benzodiazepine, diazepam, decreased social recognition and dialysate levels of ACh, while the tricyclic, imipramine, reduced social recognition and failed to enhance cholinergic transmission. In conclusion, at anxiolytic and antidepressant doses, SNAP-7941 and GW3430 improve social recognition and elevate extracellular ACh levels in FCX. This profile differentiates MCH1 receptor antagonists from conventional anxiolytic and antidepressant agents.

  12. Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex

    PubMed Central

    Wang, Chunmei; Ren, Fazheng; Hao, Yanling

    2016-01-01

    Upon exposure to exogenous pediocin-like bacteriocins, immunity proteins specifically bind to the target receptor of the mannose phosphotransferase system components (man-PTS IIC and IID), therefore preventing bacterial cell death. However, the specific recognition of immunity proteins and its associated target receptors remains poorly understood. In this study, we constructed hybrid receptors to identify the domains of IIC and/or IID recognized by the immunity protein PedB, which confers immunity to pediocin PA-1. Using Lactobacillus plantarum man-PTS EII mutant W903, the IICD components of four pediocin PA-1-sensitive strains (L. plantarum WQ0815, Leuconostoc mesenteroides 05–43, Lactobacillus salivarius REN and Lactobacillus acidophilus 05–172) were respectively co-expressed with the immunity protein PedB. Well-diffusions assays showed that only the complex formed by LpIICD from L. plantarum WQ0815 with pediocin PA-1 could be recognized by PedB. In addition, a two-step PCR approach was used to construct hybrid receptors by combining LpIIC or LpIID recognized by PedB with the other three heterologous IID or IIC compounds unrecognized by PedB, respectively. The results showed that all six hybrid receptors were recognized by pediocin PA-1. However, when IIC or IID of L. plantarum WQ0815 was replaced with any corresponding IIC or IID component from L. mesenteroides 05–43, L. salivarius REN and L. acidophilus 05–172, all the hybrid receptors could not be recognized by PedB. Taken altogether, we concluded that both IIC and IID components of the mannose phosphotransferase system play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB. PMID:27776158

  13. A novel C1qDC protein acting as pattern recognition receptor in scallop Argopecten irradians.

    PubMed

    Wang, Leilei; Wang, Lingling; Kong, Pengfei; Yang, Jialong; Zhang, Huan; Wang, Mengqiang; Zhou, Zhi; Qiu, Limei; Song, Linsheng

    2012-08-01

    The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F' as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system.

  14. Positive allosteric modulation of alpha 7 nicotinic acetylcholine receptors enhances recognition memory and cognitive flexibility in rats.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Potasiewicz, Agnieszka; Popik, Piotr

    2015-08-01

    A wide body of preclinical and clinical data suggests that alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) may represent useful targets for cognitive improvement in schizophrenia and Alzheimer׳s disease. A promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs due to their several advantages over the direct agonists. Nevertheless, the behavioural effects of this class of compounds, particularly with regard to higher-order cognitive functions, have not been broadly characterised. The aim of the present study was to evaluate the procognitive efficacies of type I and type II α7-nAChRs PAMs, N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) in the novel object recognition task (NORT), attentional set-shifting task (ASST) and five-choice serial reaction time task (5-CSRTT) in rats. Additionally, the effects of galantamine, an acetylcholinesterase inhibitor that also allosterically modulates nAChRs, were assessed. We report that CCMI (0.3-3mg/kg), PNU-120596 (0.3-3mg/kg) and galantamine (1-3mg/kg) attenuated the delay-induced impairment in NORT performance and facilitated cognitive flexibility in the ASST. Methyllycaconitine (3mg/kg) blocked the actions of CCMI, PNU-120596 and galantamine in the NORT and ASST, suggesting that the procognitive effects of these compounds are α7-nAChRs-dependent. However, none of the compounds tested affected the rats' attentional performance in the 5-CSRTT. The present findings confirm and extend the observations indicating that the positive allosteric modulation of α7-nAChRs enhances recognition memory and cognitive flexibility in preclinical tasks. Therefore, the present study supports the utility of α7-nAChRs PAMs as a potential cognitive enhancing therapy. PMID:26003081

  15. THE METABOTROPIC GLUTAMATE 2/3 RECEPTOR AGONIST LY379268 COUNTERACTED KETAMINE-AND APOMORPHINE-INDUCED PERFORMANCE DEFICITS IN THE OBJECT RECOGNITION TASK, BUT NOT OBJECT LOCATION TASK, IN RATS

    PubMed Central

    Pitsikas, Nikolaos; Markou, Athina

    2014-01-01

    Experimental evidence indicates that the non competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine and the mixed dopamine (DA) D1/D2 receptor agonist apomorphine induce schizophrenia-like symptoms in rodents, including cognitive deficits. Activation of Group II metabotropic glutamate 2/3 (mGlu2/3) receptors reduces the excessive glutamate release that is hypothesized to be associated with psychiatric disorders. Thus, mGlu2/3 receptor agonists may reverse deficits induced by excessive glutamate or DA release induced by administration of NMDA receptor antagonists and DA receptor agonists, respectively, and potentially those seen in schizophrenia. LY379268 is a selective mGlu2/3 receptor agonist that has shown to be effective in several animal models of stroke, epilepsy, and drug abuse. The present study investigated whether LY379268 antagonizes non-spatial and spatial recognition memory deficits induced by ketamine and apomorphine administration in rats. To assess the effects of the compounds on non-spatial and spatial recognition memory, the object recognition task and object location task were used. Post-training administration of LY379268 (1-3 mg/kg, i.p.) counteracted ketamine (3 mg/kg, i.p.) and apomorphine (1 mg/kg, i.p.)-induced performance deficits in the object recognition task. In contrast, LY379268 (1-3 mg/kg, i.p.) did not attenuate spatial recognition memory deficits produced by ketamine (3 mg/kg, i.p.) or apomorphine (1 mg/kg, i.p.) in the object location task. The present data show that the mGlu2/3 receptor agonist LY379268 reversed non-spatial, but not spatial, recognition memory deficits induced by NMDA receptor blockade or DA receptor agonism in rodents. Thus, such mGlu2/3 receptor agonists may be efficacious in reversing some memory deficits seen in schizophrenia patients. PMID:24859609

  16. De Novo Transcriptome Analysis Shows That SAV-3 Infection Upregulates Pattern Recognition Receptors of the Endosomal Toll-Like and RIG-I-Like Receptor Signaling Pathways in Macrophage/Dendritic Like TO-Cells

    PubMed Central

    Xu, Cheng; Evensen, Øystein; Mweemba, Hetron Munang’andu

    2016-01-01

    A fundamental step in cellular defense mechanisms is the recognition of “danger signals” made of conserved pathogen associated molecular patterns (PAMPs) expressed by invading pathogens, by host cell germ line coded pattern recognition receptors (PRRs). In this study, we used RNA-seq and the Kyoto encyclopedia of genes and genomes (KEGG) to identify PRRs together with the network pathway of differentially expressed genes (DEGs) that recognize salmonid alphavirus subtype 3 (SAV-3) infection in macrophage/dendritic like TO-cells derived from Atlantic salmon (Salmo salar L) headkidney leukocytes. Our findings show that recognition of SAV-3 in TO-cells was restricted to endosomal Toll-like receptors (TLRs) 3 and 8 together with RIG-I-like receptors (RLRs) and not the nucleotide-binding oligomerization domain-like receptors NOD-like receptor (NLRs) genes. Among the RLRs, upregulated genes included the retinoic acid inducible gene I (RIG-I), melanoma differentiation association 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). The study points to possible involvement of the tripartite motif containing 25 (TRIM25) and mitochondrial antiviral signaling protein (MAVS) in modulating RIG-I signaling being the first report that links these genes to the RLR pathway in SAV-3 infection in TO-cells. Downstream signaling suggests that both the TLR and RLR pathways use interferon (IFN) regulatory factors (IRFs) 3 and 7 to produce IFN-a2. The validity of RNA-seq data generated in this study was confirmed by quantitative real time qRT-PCR showing that genes up- or downregulated by RNA-seq were also up- or downregulated by RT-PCR. Overall, this study shows that de novo transcriptome assembly identify key receptors of the TLR and RLR sensors engaged in host pathogen interaction at cellular level. We envisage that data presented here can open a road map for future intervention strategies in SAV infection of salmon. PMID:27110808

  17. Mutations in pattern recognition receptor genes modulate seroreactivity to microbial antigens in patients with inflammatory bowel disease

    PubMed Central

    Henckaerts, Liesbet; Pierik, Marie; Joossens, Marie; Ferrante, Marc; Rutgeerts, Paul; Vermeire, Séverine

    2007-01-01

    Background and aims A number of antibodies against microbial epitopes or self‐antigens have been associated with Crohn's disease. The development of antibodies reflects a loss of tolerance to intestinal bacteria that underlies Crohn's disease, resulting in an exaggerated adaptive immune response to these bacteria. It was hypothesised that the development of antimicrobial antibodies is influenced by the presence of genetic variants in pattern recognition receptor genes. The aim of this study was therefore to investigate the influence of mutations in these innate immune receptor genes (nucleotide oligomerisation domain (NOD) 2/caspase recruitment domain (CARD) 15, NOD1/CARD4, TUCAN/CARDINAL/CARD8, Toll‐like receptor (TLR) 4, TLR2, TLR1 and TLR6) on the development of antimicrobial and antiglycan antibodies in inflammatory bowel disease (IBD). Materials and methods A cohort of 1163 unrelated patients with IBD (874 Crohn's disease, 259 ulcerative colitis, 30 indeterminate colitis) and 312 controls were analysed for anti‐Saccharomyces cerevisiae antibodies (gASCA) IgG, anti‐laminaribioside antibodies (ALCA) IgG, anti‐chitobioside antibodies (ACCA) IgA, anti‐mannobioside antibodies (AMCA) IgG and outer membrane porin (Omp) IgA and were genotyped for variants in NOD2/CARD15, TUCAN/CARDINAL/CARD8, NOD1/CARD4, TLR4, TLR1, TLR2 and TLR6. Results When compared with Crohn's disease patients without CARD15 mutations, the presence of at least one CARD15 variant in Crohn's disease patients more frequently led to gASCA positivity (66.1% versus 51.5%, p < 0.0001) and ALCA positivity (43.3% versus 34.9%, p  =  0.018) and higher gASCA titers (85.7 versus 51.8 ELISA units, p < 0.0001), independent of ileal involvement. A gene dosage effect, with increasing gASCA and ALCA positivity for patients carrying none, one and two CARD15 variants, respectively, was seen for both markers. Similarly, Crohn's disease patients carrying NOD1/CARD4 indel had a higher

  18. CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria.

    PubMed

    Yang, Jialong; Qiu, Limei; Wang, Lingling; Wei, Xiumei; Zhang, Huan; Zhang, Ying; Liu, Lin; Song, Linsheng

    2010-11-01

    Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coli, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C. farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. PMID:20659562

  19. PtLGBP, a pattern recognition receptor in Portunus trituberculatus involved in the immune response against different challenges.

    PubMed

    Chen, Y-E; Jin, S; Zhao, Q-S; Zhang, Y; Wang, C-L

    2014-09-01

    Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) is a pattern recognition receptor that can recognize and bind LPS and b-1,3-glucan. LGBP has crucial roles in innate immune defense against Gram-negative bacteria and fungi. In this study, LGBP functions in Portunus trituberculatus innate immunity were analyzed. First, the mRNA expression of PtLGBP in hemocytes, hepatopancreas, and muscle toward three typical pathogen-associated molecular patterns (PAMPs) stimulations were examined using real-time PCR. Results show that the overall trend of relative expressions of the LGBP gene in three tissues is consistent, showing up-down trend. In each group, the highest expression of the LGBP gene was at 3 and 12 h post-injection. The LGBP gene is also expressed significantly higher in the hemocytes and hepatopancreas than in the muscle. The highest level of LGBP was in the lipopolysaccharides (LPS) and glucan-injected group, whereas the lowest level was in the PGN-injected group. Furthermore, bacterial agglutination assay with polyclonal antibody specifically for PtLGBP proved that the recombinant PtLGBP (designated as rPtLGBP) could exhibit obvious agglutination activity toward Gram-negative bacteria Escherichia coli, Vibrio parahaemolyticus, and V. alginolyticus; Gram-positive bacteria Bacillus subtilis; and fungi Saccharomyces cerevisiae. LGBP in Portunus trituberculatus possibly served as a multi-functional PRR. In addition, LGBP is not only involved in the immune response against Gram-negative and fungi, as manifested in other invertebrates, but also has a significant role in anti-Gram-positive bacteria infection.

  20. Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

    PubMed

    Satoh, Tadashi; Sato, Ken; Kanoh, Akira; Yamashita, Katsuko; Yamada, Yusuke; Igarashi, Noriyuki; Kato, Ryuichi; Nakano, Akihiko; Wakatsuki, Soichi

    2006-04-14

    Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

  1. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    PubMed Central

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors. PMID:21625560

  2. Structure of Natural Killer Receptor 2B4 Bound to CD48 Reveals Basis for Heterophilic Recognition in Signaling Lymphocyte Activation Molecule Family

    SciTech Connect

    Velikovsky,C.; Deng, L.; Chlewicki, L.; Fernandez, M.; Kumar, V.; Mariuzza, R.

    2007-01-01

    Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.

  3. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    SciTech Connect

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric

    2012-02-21

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  4. Substituent directed selectivity in anion recognition by a new class of simple osmium-pyrazole derived receptors.

    PubMed

    Das, Ankita; Mondal, Prasenjit; Dasgupta, Moumita; Kishore, Nand; Lahiri, Goutam Kumar

    2016-02-14

    The present article deals with the structurally, spectroscopically and electrochemically characterised osmium-bipyridyl derived complexes [(bpy)2Os(II)(HL1)Cl]ClO4 [1]ClO4 and [(bpy)2Os(II)(HL2)Cl]ClO4 [2]ClO4 incorporating neutral and monodentate pyrazole derivatives (HL) with one free NH function (bpy = 2,2'-bipyridine, HL1 = pyrazole, HL2 = 3,5-dimethylpyrazole). The crystal structures of [1]ClO4 and [2]ClO4 reveal intramolecular hydrogen bonding interactions between the free NH proton of HL and the equatorially placed Cl(-) ligand (N-HCl) with donor-acceptor distances of 3.114(7) Å and 3.153(6) Å as well as intermolecular hydrogen bonding interactions between the NH proton and one of the oxygen atoms of ClO4(-) (N-HO) with donor-acceptor distances of 2.870(10) Å and 3.024(8) Å, respectively. The effect of hydrogen bonding interactions has translated into the less acidic nature of the NH proton of the coordinated HL with estimated pKa > 12. 1(+) and 2(+) exhibit reversible Os(II)/(III) and irreversible Os(III)/(IV) processes in CH3CN within ± 2.0 V versus SCE. The effect of 3,5-dimethyl substituted HL2 on 2(+) has been reflected in the appreciable lowering (40 mV) of the Os(II/III) potential, along with the further decrease in the acidity of the NH proton (pKa > 13.0) with regard to HL1 coordinated 1(+) (pKa: ∼ 12.3). The electronic spectral features of Os(ii) (1(+)/2(+)) and electrochemically generated Os(III) (1(2+)/2(2+)) derived complexes have been analysed by TD-DFT calculations. The efficacy of the 1(+) and 2(+) encompassing free NH proton towards the anion recognition process has been evaluated by different experimental investigations using a wide variety of anions. It however establishes that receptor 1(+) can recognise both F(-) and OAc(-) in acetonitrile solution, while 2(+) is exclusively selective for the F(-) ion. PMID:26733437

  5. Adjunctive selective estrogen receptor modulator increases neural activity in the hippocampus and inferior frontal gyrus during emotional face recognition in schizophrenia

    PubMed Central

    Ji, E; Weickert, C S; Lenroot, R; Kindler, J; Skilleter, A J; Vercammen, A; White, C; Gur, R E; Weickert, T W

    2016-01-01

    Estrogen has been implicated in the development and course of schizophrenia with most evidence suggesting a neuroprotective effect. Treatment with raloxifene, a selective estrogen receptor modulator, can reduce symptom severity, improve cognition and normalize brain activity during learning in schizophrenia. People with schizophrenia are especially impaired in the identification of negative facial emotions. The present study was designed to determine the extent to which adjunctive raloxifene treatment would alter abnormal neural activity during angry facial emotion recognition in schizophrenia. Twenty people with schizophrenia (12 men, 8 women) participated in a 13-week, randomized, double-blind, placebo-controlled, crossover trial of adjunctive raloxifene treatment (120 mg per day orally) and performed a facial emotion recognition task during functional magnetic resonance imaging after each treatment phase. Two-sample t-tests in regions of interest selected a priori were performed to assess activation differences between raloxifene and placebo conditions during the recognition of angry faces. Adjunctive raloxifene significantly increased activation in the right hippocampus and left inferior frontal gyrus compared with the placebo condition (family-wise error, P<0.05). There was no significant difference in performance accuracy or reaction time between active and placebo conditions. To the best of our knowledge, this study provides the first evidence suggesting that adjunctive raloxifene treatment changes neural activity in brain regions associated with facial emotion recognition in schizophrenia. These findings support the hypothesis that estrogen plays a modifying role in schizophrenia and shows that adjunctive raloxifene treatment may reverse abnormal neural activity during facial emotion recognition, which is relevant to impaired social functioning in men and women with schizophrenia. PMID:27138794

  6. Adjunctive selective estrogen receptor modulator increases neural activity in the hippocampus and inferior frontal gyrus during emotional face recognition in schizophrenia.

    PubMed

    Ji, E; Weickert, C S; Lenroot, R; Kindler, J; Skilleter, A J; Vercammen, A; White, C; Gur, R E; Weickert, T W

    2016-01-01

    Estrogen has been implicated in the development and course of schizophrenia with most evidence suggesting a neuroprotective effect. Treatment with raloxifene, a selective estrogen receptor modulator, can reduce symptom severity, improve cognition and normalize brain activity during learning in schizophrenia. People with schizophrenia are especially impaired in the identification of negative facial emotions. The present study was designed to determine the extent to which adjunctive raloxifene treatment would alter abnormal neural activity during angry facial emotion recognition in schizophrenia. Twenty people with schizophrenia (12 men, 8 women) participated in a 13-week, randomized, double-blind, placebo-controlled, crossover trial of adjunctive raloxifene treatment (120 mg per day orally) and performed a facial emotion recognition task during functional magnetic resonance imaging after each treatment phase. Two-sample t-tests in regions of interest selected a priori were performed to assess activation differences between raloxifene and placebo conditions during the recognition of angry faces. Adjunctive raloxifene significantly increased activation in the right hippocampus and left inferior frontal gyrus compared with the placebo condition (family-wise error, P<0.05). There was no significant difference in performance accuracy or reaction time between active and placebo conditions. To the best of our knowledge, this study provides the first evidence suggesting that adjunctive raloxifene treatment changes neural activity in brain regions associated with facial emotion recognition in schizophrenia. These findings support the hypothesis that estrogen plays a modifying role in schizophrenia and shows that adjunctive raloxifene treatment may reverse abnormal neural activity during facial emotion recognition, which is relevant to impaired social functioning in men and women with schizophrenia. PMID:27138794

  7. The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF.

    PubMed

    Haga, Arayo; Tanaka, Nobutada; Funasaka, Tatsuyoshi; Hashimoto, Kazunori; Nakamura, Kazuo T; Watanabe, Hideomi; Raz, Avraham; Nagase, Hisamitsu

    2006-05-01

    The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.

  8. Tris(triazole) tripodal receptors as selective probes for citrate anion recognition and multichannel transition and heavy metal cation sensing.

    PubMed

    González, María del Carmen; Otón, Francisco; Espinosa, Arturo; Tárraga, Alberto; Molina, Pedro

    2015-02-01

    The three-armed pyrenyl-triazole receptor 1 behaves as a highly selective fluorescent molecular sensor for citrate anions over similar carboxylates such as malate or tartrate. In addition, this receptor senses Cu(2+) cations through absorption and emission channels even in the presence of Hg(2+) metal cations. The related three-armed ferrocenyl-triazole receptor 2 behaves as a highly selective dual (redox and chromogenic) chemosensor molecule for Pb(2+) metal cations.

  9. A new tripodal receptor for molecular recognition of monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy.

    PubMed

    Vacca, Alberto; Nativi, Cristina; Cacciarini, Martina; Pergoli, Roberto; Roelens, Stefano

    2004-12-22

    A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.

  10. Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions.

    PubMed

    Reid, Delyth M; Montoya, Maria; Taylor, Philip R; Borrow, Persephone; Gordon, Siamon; Brown, Gordon D; Wong, Simon Y C

    2004-07-01

    Dectin-1 is a pathogen-recognition receptor on macrophages (MPhis), neutrophils, and dendritic cells (DCs). On MPhis and bone marrow-derived DCs, it has been shown to mediate the nonopsonic recognition of and response to soluble and particulate yeast beta-glucans. We have optimized the immunohistochemical detection of Dectin-1 and demonstrated its expression on neutrophils, subpopulations of MPhis in splenic red and white pulp, alveolar MPhis, Kupffer cells, and MPhis and DCs in the lamina propria of gut villi. This is consistent with its role in pathogen surveillance. A significant proportion of CD11c(+) splenic DCs expressed Dectin-1, but expression was not restricted to any one subset. Dectin-1 expression was low on resident MPhis and DCs of skin and was not detected on resident MPhis or DCs in kidney, heart, brain, or eye. The proposed, additional role of Dectin-1 as a coreceptor for T cell activation is supported by its expression on DCs in the T cell areas of the spleen and lymph nodes. Strong expression of Dectin-1 on subpopulations of MPhis and DCs in the medullary and corticomedullary regions of the thymus suggests a role distinct from pathogen recognition. Tissue localization thus revealed potential roles of Dectin-1 in leukocyte interactions during innate immune responses and T cell development. PMID:15107454

  11. Kin Recognition in Bacteria.

    PubMed

    Wall, Daniel

    2016-09-01

    The ability of bacteria to recognize kin provides a means to form social groups. In turn these groups can lead to cooperative behaviors that surpass the ability of the individual. Kin recognition involves specific biochemical interactions between a receptor(s) and an identification molecule(s). Recognition specificity, ensuring that nonkin are excluded and kin are included, is critical and depends on the number of loci and polymorphisms involved. After recognition and biochemical perception, the common ensuing cooperative behaviors include biofilm formation, quorum responses, development, and swarming motility. Although kin recognition is a fundamental mechanism through which cells might interact, microbiologists are only beginning to explore the topic. This review considers both molecular and theoretical aspects of bacterial kin recognition. Consideration is also given to bacterial diversity, genetic relatedness, kin selection theory, and mechanisms of recognition. PMID:27359217

  12. PWZ-029, an inverse agonist selective for α₅ GABAA receptors, improves object recognition, but not water-maze memory in normal and scopolamine-treated rats.

    PubMed

    Milić, Marija; Timić, Tamara; Joksimović, Srđan; Biawat, Poonam; Rallapalli, Sundari; Divljaković, Jovana; Radulović, Tamara; Cook, James M; Savić, Miroslav M

    2013-03-15

    Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α(5) GABA(A) inverse agonists.

  13. A C1q domain containing protein from Crassostrea gigas serves as pattern recognition receptor and opsonin with high binding affinity to LPS.

    PubMed

    Jiang, Shuai; Li, Hui; Zhang, Daoxiang; Zhang, Huan; Wang, Lingling; Sun, Jinsheng; Song, Linsheng

    2015-08-01

    C1q proteins serve as pattern recognition receptors and involve in the pathogen recognition and complement pathway activation. In the present study, a novel C1q domain containing protein from Crassostrea gigas (designated CgC1qDC-1) was isolated by liposaccharide-Sepharose 6B affinity chromatography. The coding sequence of CgC1qDC-1 gene was determined by performing a homologous search of eight tryptic peptides identified by MALDI-TOF/TOF-MS against the genome of C. gigas. The coding sequence of CgC1qDC-1 was of 387 bp encoding a polypeptide of 128 amino acids containing a typical globular C1q domain. The globular C1q domain possessed eight β strands with a jelly-roll topology structure, which was similar to the structure of human gC1q domain. The mRNA transcripts of CgC1qDC-1 were dominantly expressed in mantle and hemocytes, while low expressed in hepatopancreas, gonad, gill and muscle. The expression level of CgC1qDC-1 increased drastically at 6 h after Vibrio splendidus stimulation, and then gradually fell to the normal level at about 24 h. ELISA assay quantified that CgC1qDC-1 bound to LPS with high binding affinity (Kd = 0.09 × 10(-6) M). Moreover, CgC1qDC-1 significantly enhanced the phagocytosis of oyster hemocytes towards Gram-negative bacteria Escherichia coli and V. splendidus. These results collectively indicated that CgC1qDC-1 could serve as pattern recognition receptor and opsonin in the innate immune response against invading Gram-negative bacteria.

  14. Distinct Conformations of Ly49 Natural Killer Cell Receptors Mediate MHC Class I Recognition in Trans and Cis

    SciTech Connect

    Back, J.; Malchiodi, E; Cho, S; Scarpellino, L; Schneider, P; Kerzic, M; Mariuzza, R; Held, W

    2009-01-01

    Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.

  15. Oestrogen and Progesterone Receptors and COX-2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama).

    PubMed

    Bianchi, C P; Meikle, A; Benavente, M A; Álvarez, M A; Trasorras, V L; Miragaya, M H; Rodríguez, E; Aba, M A

    2015-12-01

    Endometrial expression of oestrogen receptor-α (ERα), progesterone receptor (PR) and cyclooxigenase-2 (COX-2) was evaluated in non-pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non-pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post-induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post-mating and by progesterone profile on day 12 post-mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX-2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non-pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post-induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX-2 expression was lower in pregnant than in non-pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF(2α) secretion during early pregnancy by decreasing the endometrial expression of COX-2 in the luminal epithelium of pregnant llamas. PMID:26446171

  16. Oestrogen and Progesterone Receptors and COX-2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama).

    PubMed

    Bianchi, C P; Meikle, A; Benavente, M A; Álvarez, M A; Trasorras, V L; Miragaya, M H; Rodríguez, E; Aba, M A

    2015-12-01

    Endometrial expression of oestrogen receptor-α (ERα), progesterone receptor (PR) and cyclooxigenase-2 (COX-2) was evaluated in non-pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non-pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post-induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post-mating and by progesterone profile on day 12 post-mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX-2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non-pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post-induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX-2 expression was lower in pregnant than in non-pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF(2α) secretion during early pregnancy by decreasing the endometrial expression of COX-2 in the luminal epithelium of pregnant llamas.

  17. Bordetella pertussis Lipid A Recognition by Toll-like Receptor 4 and MD-2 Is Dependent on Distinct Charged and Uncharged Interfaces.

    PubMed

    Maeshima, Nina; Evans-Atkinson, Tara; Hajjar, Adeline M; Fernandez, Rachel C

    2015-05-22

    Lipid A in LPS activates innate immunity through the Toll-like receptor 4 (TLR4)-MD-2 complex on host cells. Variation in lipid A has significant consequences for TLR4 activation and thus may be a means by which Gram-negative bacteria modulate host immunity. However, although even minor changes in lipid A structure have been shown to affect downstream immune responses, the mechanism by which the TLR4-MD-2 receptor complex recognizes these changes is not well understood. We previously showed that strain BP338 of the human pathogen Bordetella pertussis, the causative agent of whooping cough, modifies its lipid A by the addition of glucosamine moieties that promote TLR4 activation in human, but not mouse, macrophages. Using site-directed mutagenesis and an NFκB reporter assay screen, we have identified several charged amino acid residues in TLR4 and MD-2 that are important for these species-specific responses; some of these are novel for responses to penta-acyl B. pertussis LPS, and their mutation does not affect the response to hexa-acylated Escherichia coli LPS or tetra-acylated lipid IVA. We additionally show evidence that suggests that recognition of penta-acylated B. pertussis lipid A is dependent on uncharged amino acids in TLR4 and MD-2 and that this is true for both human and mouse TLR4-MD-2 receptors. Taken together, we have demonstrated that the TLR4-MD-2 receptor complex recognizes variation in lipid A molecules using multiple sites for receptor-ligand interaction and propose that host-specific immunity to a particular Gram-negative bacterium is, at least in part, mediated by very subtle tuning of one of the earliest interactions at the host-pathogen interface.

  18. Evolutionarily conserved recognition and innate immunity to fungal pathogens by the scavenger receptors SCARF1 and CD36

    PubMed Central

    Mylonakis, Eleftherios; Tampakakis, Emmanouil; Colvin, Richard A.; Seung, Edward; Puckett, Lindsay; Tai, Melissa F.; Stewart, Cameron R.; Pukkila-Worley, Read; Hickman, Suzanne E.; Moore, Kathryn J.; Calderwood, Stephen B.; Hacohen, Nir; Luster, Andrew D.; El Khoury, Joseph

    2009-01-01

    Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was β-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are β-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens. PMID:19237602

  19. The Arabidopsis LecRK-VI.2 associates with the pattern-recognition receptor FLS2 and primes Nicotiana benthamiana pattern-triggered immunity.

    PubMed

    Huang, Pin-Yao; Yeh, Yu-Hung; Liu, An-Chi; Cheng, Chiu-Ping; Zimmerli, Laurent

    2014-07-01

    Pattern-triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad-spectrum disease resistance. PTI is activated upon perception of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs). We have recently demonstrated that the L-type lectin receptor kinase-VI.2 (LecRK-VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull-down, bimolecular fluorescence complementation and co-immunoprecipitation analyses that LecRK-VI.2 associates with the PRR FLS2. We also demonstrated that LecRK-VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK-VI.2 were indeed more resistant to virulent hemi-biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK-VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK-VI.2 in N. benthamiana primed PTI-mediated reactive oxygen species production, mitogen-activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK-VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.

  20. The role of pattern-recognition receptors in graft-versus-host disease and graft-versus-leukemia after allogeneic stem cell transplantation.

    PubMed

    Heidegger, Simon; van den Brink, Marcel R M; Haas, Tobias; Poeck, Hendrik

    2014-01-01

    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only treatment with curative potential for certain aggressive hematopoietic malignancies. Its success is limited by acute graft-versus-host disease (GVHD), a life-threatening complication that occurs when allo-reactive donor T cells attack recipient organs. There is growing evidence that microbes and innate pattern-recognition receptors (PRRs) such as toll-like receptors (TLR) and nod-like receptors (NLR) are critically involved in the pathogenesis of acute GVHD. Currently, a widely accepted model postulates that intensive chemotherapy and/or total-body irradiation during pre-transplant conditioning results in tissue damage and a loss of epithelial barrier function. Subsequent translocation of bacterial components as well as release of endogenous danger molecules stimulate PRRs of host antigen-presenting cells to trigger the production of pro-inflammatory cytokines (cytokine storm) that modulate T cell allo-reactivity against host tissues, but eventually also the beneficial graft-versus-leukemia (GVL) effect. Given the limitations of existing immunosuppressive therapies, a better understanding of the molecular mechanisms that govern GVHD versus GVL is urgently needed. This may ultimately allow to design modulators, which protect from GvHD but preserve donor T-cell attack on hematologic malignancies. Here, we will briefly summarize current knowledge about the role of innate immunity in the pathogenesis of GVHD and GVL following allo-HSCT.

  1. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation.

    PubMed

    Steinbrecher, U P; Lougheed, M; Kwan, W C; Dirks, M

    1989-09-15

    Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of

  2. Differential recognition by CD28 of its cognate counter receptors CD80 (B7.1) and B70 (B7.2): analysis by site directed mutagenesis.

    PubMed

    Truneh, A; Reddy, M; Ryan, P; Lyn, S D; Eichman, C; Couez, D; Hurle, M R; Sekaly, R P; Olive, D; Sweet, R

    1996-02-01

    CD28, which is a member of the immunoglobulin superfamily of molecules (IgSF), is a homodimer of two polypeptides containing a single V-like domain with short transmembrane and cytoplasmic regions. It serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and B70, expressed on antigen presenting cells. In the current study, we investigated the regions of CD28 which are involved in its interactions with CD80 and B70, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. Truncation or substitution of a stretch of a proline rich "hallmark" sequence, "MYPPPY", abrogates binding to CD80 or B70, while retaining CD28 mAb epitopes and cell surface expression. On an Ig-fold model of the CD28 V-domain, this fully conserved motif localizes to a CDR3-like region. Mutations introduced into other loops, including the CDRI-like and CDR2-like regions, had very little effect on CD80 or B70 binding. Mutations introduced within the predicted beta-strand regions caused loss of receptor expression. Conservative substitution of both the flanking tyrosine residues within the "MYPPPY" motif with phenylalanine, caused loss of binding to B70 but not to CD80. These results show that, although the same overall region on CD28 may be involved in the interactions with CD80 and B70, subtle but important differences distinguish recognition by the two molecules. These finding, along with previous observations on the differential pattern of expression and tissue distribution of CD80 and B70, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.

  3. Hemispherand-Strapped Calix[4]pyrrole: An Ion-pair Receptor for the Recognition and Extraction of Lithium Nitrite.

    PubMed

    He, Qing; Zhang, Zhan; Brewster, James T; Lynch, Vincent M; Kim, Sung Kuk; Sessler, Jonathan L

    2016-08-10

    The hemispherand-strapped calix[4]pyrrole (1) acts as an ion pair receptor that exhibits selectivity for lithium salts. In organic media (CD2Cl2 and CD3OD, v/v, 9:1), receptor 1 binds LiCl with high preference relative to NaCl, KCl, and RbCl. DFT calculations provided support for the observed selectivity. Single crystal structures of five different lithium ion-pair complexes of 1 were obtained. In the case of LiCl, a single bridging water molecule between the lithium cation and chloride anion was observed, while tight contact ion pairs were observed in the case of the LiBr, LiI, LiNO3, and LiNO2 salts. Receptor 1 proved effective as an extractant for LiNO2 under both model solid-liquid and liquid-liquid extraction conditions. PMID:27442768

  4. Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.

    PubMed

    Burg, John S; Ingram, Jessica R; Venkatakrishnan, A J; Jude, Kevin M; Dukkipati, Abhiram; Feinberg, Evan N; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O; Ploegh, Hidde L; Garcia, K Christopher

    2015-03-01

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state. PMID:25745166

  5. Xa21D encodes a receptor-like molecule with a leucine-rich repeat domain that determines race-specific recognition and is subject to adaptive evolution.

    PubMed Central

    Wang, G L; Ruan, D L; Song, W Y; Sideris, S; Chen, L; Pi, L Y; Zhang, S; Zhang, Z; Fauquet, C; Gaut, B S; Whalen, M C; Ronald, P C

    1998-01-01

    The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition. PMID:9596635

  6. Pattern recognition receptor mediated downregulation of microRNA‐650 fine‐tunes MxA expression in dendritic cells infected with influenza A virus

    PubMed Central

    Khatamzas, Elham; Liu, Xiao; Brain, Oliver; Delmiro Garcia, Magno; Leslie, Alasdair; Danis, Benedicte; Mayer, Alice; Baban, Dilair; Ragoussis, Jiannis; Weber, Alexander N. R.; Simmons, Alison

    2015-01-01

    MicroRNAs are important posttranscriptional regulators of gene expression, which have been shown to fine‐tune innate immune responses downstream of pattern recognition receptor (PRR) signaling. This study identifies miR‐650 as a novel PRR‐responsive microRNA that is downregulated upon stimulation of primary human monocyte‐derived dendritic cells (MDDCs) with a variety of different microbe‐associated molecular patterns. A comprehensive target search combining in silico analysis, transcriptional profiling, and reporter assays reveals that miR‐650 regulates several well‐known interferon‐stimulated genes, including IFIT2 and MXA. In particular, downregulation of miR‐650 in influenza A infected MDDCs enhances the expression of MxA and may therefore contribute to the establishment of an antiviral state. Together these findings reveal a novel link between miR‐650 and the innate immune response in human MDDCs. PMID:26460926

  7. Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition.

    PubMed

    Alvarez-Domínguez, C; Vázquez-Boland, J A; Carrasco-Marín, E; López-Mato, P; Leyva-Cobián, F

    1997-01-01

    The mechanisms by which the intracellular pathogen Listeria monocytogenes interacts with the host cell surface remain largely unknown. In this study, we investigated the role of heparan sulfate proteoglycans (HSPG) in listerial infection. Pretreatment of bacteria with heparin or heparan sulfate (HS), but not with other glycosaminoglycans, inhibited attachment and subsequent uptake by IC-21 murine macrophages and CHO epithelial-like cells. Specific removal of HS from target cells with heparinase III significantly impaired listerial adhesion and invasion. Mutant CHO cells deficient in HS synthesis bound and internalized significantly fewer bacteria than wild-type cells did. Pretreatment of target cells with the HS-binding proteins fibronectin and platelet factor 4, or with heparinase III, impaired listerial infectivity only in those cells expressing HS. Moreover, a synthetic peptide corresponding to the HS-binding ligand in Plasmodium falciparum circumsporozoite protein (pepPf1) inhibited listerial attachment to IC-21 and CHO cells. A motif very similar to the HS-binding site of pepPf1 was found in the N-terminal region of ActA, the L. monocytogenes surface protein responsible for actin-based bacterial motility and cell-to-cell spread. In the same region of ActA, several clusters of positively charged amino acids which could function as HS-binding domains were identified. An ActA-deficient mutant was significantly impaired in attachment and entry due to altered HS recognition functions. This work shows that specific interaction with an HSPG receptor present on the surface of both professional and nonprofessional phagocytes is involved in L. monocytogenes cytoadhesion and invasion and strongly suggests that the bacterial surface protein ActA may be a ligand mediating HSPG receptor recognition. PMID:8975895

  8. Gene polymorphisms of Toll-like and related recognition receptors in relation to the vaginal carriage of Gardnerella vaginalis and Atopobium vaginae.

    PubMed

    Verstraelen, Hans; Verhelst, Rita; Nuytinck, Lieve; Roelens, Kristien; De Meester, Els; De Vos, Daniel; Van Thielen, Martine; Rossau, Rudi; Delva, Wim; De Backer, Ellen; Vaneechoutte, Mario; Temmerman, Marleen

    2009-01-01

    Host genetic factors have previously been found to act as determinants of differential susceptibility to major infectious diseases. It is less clear whether such polymorphisms may also impose on pathogen recognition in mucosal overgrowth conditions such as bacterial vaginosis, an anaerobic overgrowth condition characterised by the presence of a vaginal biofilm consisting of the Gram-positive anaerobes Gardnerella vaginalis and Atopobium vaginae. We selected 34 single nucleotide polymorphisms pertaining to 9 genes involved with Toll-like receptor-mediated pathogen recognition and/or regulation (LBP, CD14, TLR1, TLR2, TLR4, TLR6, MD2, CARD15 and SIGIRR) and assessed in a nested case-control study their putative association with bacterial vaginosis, as diagnosed by Gram staining, and with the vaginal carriage of A. vaginae and G. vaginalis, as determined by species-specific PCR, among 144 pregnant women. Carriage of G. vaginalis during early pregnancy was associated with the -1155A>G substitution in the promoter region of the MD2 gene (p=0.041). The presence of A. vaginae during the first half of the pregnancy was significantly associated with the CD14 intron 2 1342G>T (p=0.039), the TLR1 exon 4 743A>G (p=0.038), and the CARD15 exon 4 14772A>T (p=0.012) polymorphisms, and marginally significantly associated with the LBP exon13 26842C>T (p=0.056), the CD14 promoter -260C>T (p=0.052), and the TLR1 promoter -7202A>G (p=0.062) polymorphisms. However, no association between gene polymorphisms and bacterial vaginosis as such could be documented. Our data suggest that some degree of genetic susceptibility involving pathogen recognition may occur with the key bacterial vaginosis organism, A. vaginae.

  9. Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition.

    PubMed

    De Oliveira, D B; Harfouch-Hammoud, E; Otto, H; Papandreou, N A; Stern, L J; Cohen, H; Boehm, B O; Bach, J; Caillat-Zucman, S; Walk, T; Jung, G; Eliopoulos, E; Papadopoulos, G K; van Endert, P M

    2000-10-01

    Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.

  10. Involvement of a versatile pattern recognition receptor, apolipophorin-III in prophenoloxidase activation and antibacterial defense of the Chinese oak silkworm, Antheraea pernyi.

    PubMed

    Wen, Daihua; Wang, Xialu; Shang, Lei; Huang, Yu; Li, Tienan; Wu, Chunfu; Zhang, Rong; Zhang, Jinghai

    2016-12-01

    Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi. PMID:27387151

  11. Analysis of a Foot-and-Mouth Disease Virus Type A24 Isolate Containing an SGD Receptor Recognition Site In Vitro and Its Pathogenesis in Cattle

    PubMed Central

    Rieder, Elizabeth; Henry, Tina; Duque, Hernando; Baxt, Barry

    2005-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via an Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. Following serial passages of a type A24 Cruzeiro virus (A24Cru) in bovine, via tongue inoculation, a virus was generated which contained an SGD sequence in the cell receptor-binding site and expressed a turbid plaque phenotype in BHK-21 cells. Propagation of this virus in these cells resulted in the rapid selection of viruses that grew to higher titers, produced clear plaques, and now contained an RGD sequence in place of the original SGD. To study the role of the SGD sequence in FMDV receptor recognition and bovine virulence, we assembled an infectious cDNA clone of an RGD-containing A24Cru and derived mutant clones containing either SGD with a single nucleotide substitution in the R144 codon or double substitutions at this position to prevent mutation of the S to an R. The SGD viruses grew poorly in BHK-21 cells and stably maintained the sequence during propagation in BHK-21 cells expressing the bovine αVβ6 integrin (BHK3-αVβ6), as well as in experimentally infected and contact steers. While all the SGD-containing viruses used only the bovine αVβ6 integrin as a cellular receptor with relatively high efficiency, the revertant RGD viruses utilized either the αVβ1 or αVβ3 bovine integrins with higher efficiency than αVβ6 and grew well in BHK-21 cells. Replacing the R at the −1 SGD position with either K or E showed that this residue did not contribute to integrin utilization in vitro. These results illustrate the rapid evolution of FMDV with alteration in receptor specificity and suggest that viruses with sequences other than RGD, but closely related to it, can still infect via integrin receptors and induce and transmit the disease to susceptible animals. PMID:16189001

  12. Identification of Novel Small-Molecule Agonists for Human Formyl Peptide Receptors and Pharmacophore Models of Their RecognitionS⃞

    PubMed Central

    Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Schepetkin, Igor A.; Ye, Richard D.; Rabiet, Marie-Josèphe; Jutila, Mark A.

    2010-01-01

    N-formyl peptide receptor (FPR1) and N-formyl peptide receptor-like 1 (FPRL1, now known as FPR2) are G protein-coupled receptors involved in host defense and sensing cellular dysfunction. Because of the potential for FPR1/FPR2 as a therapeutic target, our recent high-throughput screening efforts have focused on the identification of unique nonpeptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug-like molecules for their ability to induce intracellular calcium mobilization in RBL-2H3 cells transfected with human FPR1 or FPR2. Screening of these compounds resulted in the identification of novel and potent agonists that activated both FPR1 and FPR2, as well as compounds that were specific for either FPR1 or FPR2 with EC50 values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL-60 cells transfected with human FPR1 and FPR2. In addition, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils, irrespective of agonist specificity for FPR1 or FPR2. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N-formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of their ligand/receptor interaction. PMID:19903830

  13. Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C.

    PubMed

    Sullivan, Lucy C; Berry, Richard; Sosnin, Natasha; Widjaja, Jacqueline M L; Deuss, Felix A; Balaji, Gautham R; LaGruta, Nicole L; Mirams, Michiko; Trapani, Joseph A; Rossjohn, Jamie; Brooks, Andrew G; Andrews, Daniel M

    2016-09-01

    Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 μm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 μm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated β2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C β4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules. PMID:27385590

  14. IL-22 is rapidly induced by Pathogen Recognition Receptors Stimulation in Bone-Marrow-derived Dendritic Cells in the Absence of IL-23

    PubMed Central

    Fumagalli, Silvia; Torri, Anna; Papagna, Angela; Citterio, Stefania; Mainoldi, Federica; Foti, Maria

    2016-01-01

    In vertebrates, microorganisms are recognized by pathogen recognition receptors (PRRs). Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of genes. Within these genes, the cytokines family plays a crucial function because of its role in adaptive immunity induction and in tissue-specific functional regulation, such as tissue repair and tissue homeostasis during steady state conditions. Within the myeloid compartment, dendritic cells (DCs) release a variety of inflammatory cytokines in response to microbes. In this study, we show that BMDCs release IL-22 directly upon PRRs activation without the need of IL-23 signaling as reported for other IL22-producing cells. Moreover, we demonstrate that cytokine IL-22 is rapidly released in a cell-specific manner as macrophages are not able to produce IL-22 through the same PRRs system. In addition, we characterize the intracellular signaling cascade required for IL-22 release in BMDCs. Myd88, MEK1/2, NFkb and AhR, but not p38, NFAT, and RORgt, were found to be involved in IL-22 regulation in DCs. Our study suggests that BMDCs possess a unique intracellular molecular plasticity which, once activated, directs different BMDCs functions in a cell-specific manner. PMID:27652524

  15. Structure-Based Mutagenesis of the Substrate-Recognition Domain of Nrdp1/FLRF Identifies the Binding Site for the Receptor Tyrosine Kinase ErbB3

    SciTech Connect

    Bouyain,S.; Leahy, D.

    2007-01-01

    The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.

  16. A TNFSF15 disease-risk polymorphism increases pattern-recognition receptor-induced signaling through caspase-8-induced IL-1.

    PubMed

    Hedl, Matija; Abraham, Clara

    2014-09-16

    Inflammatory diseases are characterized by dysregulated cytokine production. Altered functions for most risk loci, including the inflammatory bowel disease and leprosy-associated tumor necrosis factor ligand superfamily member 15 (TNFSF15) region, are unclear. Regulation of pattern-recognition-receptor (PRR)-induced signaling and cytokines is crucial for immune homeostasis; TNFSF15:death receptor 3 (DR3) contributions to PRR responses have not been described. We found that human macrophages expressed DR3 and that TNFSF15:DR3 interactions were critical for amplifying PRR-initiated MAPK/NF-κB/PI3K signaling and cytokine secretion in macrophages. Mechanisms mediating TNFSF15:DR3 contributions to PRR outcomes included TACE-induced TNFSF15 cleavage to soluble TNFSF15; soluble TNFSF15 then led to TRADD/FADD/MALT-1- and caspase-8-mediated autocrine IL-1 secretion. Notably, TNFSF15 treatment also induced cytokine secretion through a caspase-8-dependent pathway in intestinal myeloid cells. Importantly, rs6478108 A disease risk-carrier macrophages demonstrated increased TNFSF15 expression and PRR-induced signaling and cytokines. Taken together, TNFSF15:DR3 interactions amplify PRR-induced signaling and cytokines, and the rs6478108 TNFSF15 disease-risk polymorphism results in a gain of function.

  17. Subtle Changes in Peptide Conformation Profoundly Affect Recognition of the Non-Classical MHC Class I Molecule HLA-E by the CD94-NKG2 Natural Killer Cell Receptors

    SciTech Connect

    Hoare, Hilary L; Sullivan, Lucy C; Clements, Craig S; Ely, Lauren K; Beddoe, Travis; Henderson, Kate N; Lin, Jie; Reid, Hugh H; Brooks, Andrew G; Rossjohn, Jamie

    2008-03-31

    Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I molecule that binds peptides derived from the leader sequences of other HLA class I molecules. Natural killer cell recognition of these HLA-E molecules, via the CD94-NKG2 natural killer family, represents a central innate mechanism for monitoring major histocompatibility complex expression levels within a cell. The leader sequence-derived peptides bound to HLA-E exhibit very limited polymorphism, yet subtle differences affect the recognition of HLA-E by the CD94-NKG2 receptors. To better understand the basis for this peptide-specific recognition, we determined the structure of HLA-E in complex with two leader peptides, namely, HLA-Cw*07 (VMAPRALLL), which is poorly recognised by CD94-NKG2 receptors, and HLA-G*01 (VMAPRTLFL), a high-affinity ligand of CD94-NKG2 receptors. A comparison of these structures, both of which were determined to 2.5-Å resolution, revealed that allotypic variations in the bound leader sequences do not result in conformational changes in the HLA-E heavy chain, although subtle changes in the conformation of the peptide within the binding groove of HLA-E were evident. Accordingly, our data indicate that the CD94-NKG2 receptors interact with HLA-E in a manner that maximises the ability of the receptors to discriminate between subtle changes in both the sequence and conformation of peptides bound to HLA-E.

  18. A lysine-rich motif in the phosphatidylserine receptor PSR-1 mediates recognition and removal of apoptotic cells

    PubMed Central

    Yang, Hengwen; Chen, Yu-Zen; Zhang, Yi; Wang, Xiaohui; Zhao, Xiang; Godfroy, James I.; Liang, Qian; Zhang, Man; Zhang, Tianying; Yuan, Quan; Royal, Mary Ann; Driscoll, Monica; Xia, Ning-Shao; Yin, Hang; Xue, Ding

    2014-01-01

    The conserved phosphatidylserine receptor (PSR) was first identified as a receptor for phosphatidylserine, an "eat-me" signal exposed by apoptotic cells. However, several studies suggest that PSR may also act as an arginine demethylase, a lysyl hydroxylase, or an RNA binding protein through its N-terminal JmjC domain. How PSR might execute drastically different biochemical activities, and whether they are physiologically significant, remain unclear. Here we report that a lysine-rich motif in the extracellular domain of PSR-1, the Caenorhabditis elegans PSR, mediates specific phosphatidylserine binding in vitro and clearance of apoptotic cells in vivo. This motif also mediates phosphatidylserine-induced oligomerization of PSR-1, suggesting a mechanism by which PSR-1 activates phagocytosis. Mutations in the phosphatidylserine-binding motif, but not in its Fe(II) binding site critical for the JmjC activity, abolish PSR-1 phagocytic function. Moreover, PSR-1 enriches and clusters around apoptotic cells during apoptosis. These results establish that PSR-1 is a conserved, phosphatidylserine-recognizing phagocyte receptor. PMID:25564762

  19. Promiscuous presentation and recognition of nucleosomal autoepitopes in lupus: role of autoimmune T cell receptor alpha chain.

    PubMed

    Shi, Y; Kaliyaperumal, A; Lu, L; Southwood, S; Sette, A; Michaels, M A; Datta, S K

    1998-02-01

    T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).

  20. Blonanserin Ameliorates Phencyclidine-Induced Visual-Recognition Memory Deficits: the Complex Mechanism of Blonanserin Action Involving D3-5-HT2A and D1-NMDA Receptors in the mPFC

    PubMed Central

    Hida, Hirotake; Mouri, Akihiro; Mori, Kentaro; Matsumoto, Yurie; Seki, Takeshi; Taniguchi, Masayuki; Yamada, Kiyofumi; Iwamoto, Kunihiro; Ozaki, Norio; Nabeshima, Toshitaka; Noda, Yukihiro

    2015-01-01

    Blonanserin differs from currently used serotonin 5-HT2A/dopamine-D2 receptor antagonists in that it exhibits higher affinity for dopamine-D2/3 receptors than for serotonin 5-HT2A receptors. We investigated the involvement of dopamine-D3 receptors in the effects of blonanserin on cognitive impairment in an animal model of schizophrenia. We also sought to elucidate the molecular mechanism underlying this involvement. Blonanserin, as well as olanzapine, significantly ameliorated phencyclidine (PCP)-induced impairment of visual-recognition memory, as demonstrated by the novel-object recognition test (NORT) and increased extracellular dopamine levels in the medial prefrontal cortex (mPFC). With blonanserin, both of these effects were antagonized by DOI (a serotonin 5-HT2A receptor agonist) and 7-OH-DPAT (a dopamine-D3 receptor agonist), whereas the effects of olanzapine were antagonized by DOI but not by 7-OH-DPAT. The ameliorating effect was also antagonized by SCH23390 (a dopamine-D1 receptor antagonist) and H-89 (a protein kinase A (PKA) inhibitor). Blonanserin significantly remediated the decrease in phosphorylation levels of PKA at Thr197 and of NR1 (an essential subunit of N-methyl-D-aspartate (NMDA) receptors) at Ser897 by PKA in the mPFC after a NORT training session in the PCP-administered mice. There were no differences in the levels of NR1 phosphorylated at Ser896 by PKC in any group. These results suggest that the ameliorating effect of blonanserin on PCP-induced cognitive impairment is associated with indirect functional stimulation of the dopamine-D1-PKA-NMDA receptor pathway following augmentation of dopaminergic neurotransmission due to inhibition of both dopamine-D3 and serotonin 5-HT2A receptors in the mPFC. PMID:25120077

  1. Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile.

    PubMed

    Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N; Klassen, John S; Ng, Kenneth K S

    2014-01-24

    Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.

  2. pH-dependent recognition of apoptotic and necrotic cells by the human dendritic cell receptor DEC205

    PubMed Central

    Cao, Longxing; Shi, Xiangyi; Chang, Haishuang; Zhang, Qinfen; He, Yongning

    2015-01-01

    Dendritic cells play important roles in regulating innate and adaptive immune responses. DEC205 (CD205) is one of the major endocytotic receptors on dendritic cells and has been widely used for vaccine generation against viruses and tumors. However, little is known about its structure and functional mechanism. Here we determine the structure of the human DEC205 ectodomain by cryoelectron microscopy. The structure shows that the 12 extracellular domains form a compact double ring-shaped conformation at acidic pH and become extended at basic pH. Biochemical data indicate that the pH-dependent conformational change of DEC205 is correlated with ligand binding and release. DEC205 only binds to apoptotic and necrotic cells at acidic pH, whereas live cells cannot be recognized by DEC205 at either acidic or basic conditions. These results suggest that DEC205 is an immune receptor that recognizes apoptotic and necrotic cells specifically through a pH-dependent mechanism. PMID:26039988

  3. Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

    SciTech Connect

    Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

    2009-01-01

    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

  4. Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile*

    PubMed Central

    Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N.; Klassen, John S.; Ng, Kenneth K. S.

    2014-01-01

    Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents. PMID:24311789

  5. pH-Dependent recognition of apoptotic and necrotic cells by the human dendritic cell receptor DEC205.

    PubMed

    Cao, Longxing; Shi, Xiangyi; Chang, Haishuang; Zhang, Qinfen; He, Yongning

    2015-06-01

    Dendritic cells play important roles in regulating innate and adaptive immune responses. DEC205 (CD205) is one of the major endocytotic receptors on dendritic cells and has been widely used for vaccine generation against viruses and tumors. However, little is known about its structure and functional mechanism. Here we determine the structure of the human DEC205 ectodomain by cryoelectron microscopy. The structure shows that the 12 extracellular domains form a compact double ring-shaped conformation at acidic pH and become extended at basic pH. Biochemical data indicate that the pH-dependent conformational change of DEC205 is correlated with ligand binding and release. DEC205 only binds to apoptotic and necrotic cells at acidic pH, whereas live cells cannot be recognized by DEC205 at either acidic or basic conditions. These results suggest that DEC205 is an immune receptor that recognizes apoptotic and necrotic cells specifically through a pH-dependent mechanism.

  6. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

    PubMed

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  7. Tracing the evolutionary lineage of pattern recognition receptor homologues in vertebrates: An insight into reptilian immunity via de novo sequencing of the wall lizard splenic transcriptome.

    PubMed

    Priyam, Manisha; Tripathy, Mamta; Rai, Umesh; Ghorai, Soma Mondal

    2016-04-01

    Reptiles remain a deprived class in the area of genomic and molecular resources for the vertebrate classes. The transition of squamates from aquatic to terrestrial mode of life caused profound changes in their immune system to combat the altered variety of pathogens on land. The current study aims at delineating the evolution of defence mechanisms in wall lizard, Hemidactylus flaviviridis, by exploring its immunome. De novo sequencing of splenic transcriptome from wall lizard on the Illumina Hi-Seq platform generated 258,128 unique transcripts with an average GC content of 45%. Annotation of 555,557 and 6812 transcripts was carried out against NCBI (non-redundant database) and UniProt databases, respectively. The KEGG pathway annotation of transcripts classified them into 39 processes of six pathway function categories. A total of 3824 transcripts, involved in 23 immune-related pathways, were identified in the immune-relevant cluster built by harvesting the genes under KEGG pathways of immune system and immune diseases. Forty-two percent of the immune-relevant cluster was represented by pattern-recognition receptors (PRRs), of which the maximum number of transcripts was attributed to the Toll-like receptor (TLR) signalling pathway. Nine PRRs with potential full-length coding sequences were sorted for phylogenetic analysis and comparative domain analysis across the vertebrate lineage. They included DEC205/lymphocyte antigen 75 (ly75), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NOD-like receptor family CARD domain-containing 3 (NLRC3), nucleotide-binding oligomerisation domain, leucine-rich repeat-containing X1 (NLRX1), DDX58/retinoic acid-inducible gene 1 (RIG-1), Toll-like receptor 3 (TLR3), TLR4, TLR5 and TLR7. From selection studies of these genes, we inferred positive selection for ly75, NOD1, RIG-1, TLR3 and TLR4. Apart from contributing to the scarce genomic resources available for reptiles and giving a broad scope for the immune

  8. Beta-adrenergic receptors link NO/sGC/PKG signaling to BDNF expression during the consolidation of object recognition long-term memory.

    PubMed

    Furini, Cristiane R; Rossato, Janine I; Bitencourt, Lucas L; Medina, Jorge H; Izquierdo, Iván; Cammarota, Martín

    2010-05-01

    The nitric oxide (NO)/soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway is important for memory processing, but the identity of its downstream effectors as well as its actual participation in the consolidation of nonaversive declarative long-term memory (LTM) remain unknown. Here, we show that training rats in an object recognition (OR) learning task rapidly increased nitrites/nitrates (NOx) content in the CA1 region of the dorsal hippocampus while posttraining intra-CA1 microinfusion of the neuronal NO synthase (nNOS) inhibitor L-NN hindered OR LTM retention without affecting memory retrieval or other behavioral variables. The amnesic effect of L-NN was not state dependent, was mimicked by the sGC inhibitor LY83583 and the PKG inhibitor KT-5823, and reversed by coinfusion of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the PKG activator 8-bromoguanosine 3',5'-cyclic monophosphate (8Br-cGMP). SNAP did not affect the amnesic effect of LY83583 and KT-5823. Conversely, 8Br-cGMP overturned the amnesia induced by LY83583 but not that caused by KT-5823. Intra-CA1 infusion of the beta-adrenergic receptor blocker timolol right after training hindered OR LTM and, although coadministration of noradrenaline reversed the amnesia caused by L-NN, LY83583, and KT5823, the amnesic effect of timolol was unaffected by coinfusion of 8Br-cGMP or SNAP, indicating that hippocampal beta-adrenergic receptors act downstream NO/sGC/PKG signaling. We also found that posttraining intra-CA1 infusion of function-blocking anti-brain-derived neurotrophic factor (BDNF) antibodies hampered OR LTM retention, whereas OR training increased CA1 BDNF levels in a nNOS- and beta-adrenergic receptor-dependent manner. Taken together, our results demonstrate that NO/sGC/PKG signaling in the hippocampus is essential for OR memory consolidation and suggest that beta-adrenergic receptors link the activation of this pathway to BDNF expression during the consolidation of declarative

  9. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    NASA Astrophysics Data System (ADS)

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-04-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds.

  10. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    PubMed Central

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-01-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds. PMID:27075233

  11. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.

  12. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.

    PubMed

    Ozinsky, A; Underhill, D M; Fontenot, J D; Hajjar, A M; Smith, K D; Wilson, C B; Schroeder, L; Aderem, A

    2000-12-01

    Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cell-wall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-alpha production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-alpha production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature. PMID:11095740

  13. Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

    2011-01-01

    Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

  14. The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

    PubMed Central

    Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

    2016-01-01

    Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

  15. The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites.

    PubMed

    Neufeldt, Christopher J; Joyce, Michael A; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale, Michael; Tyrrell, D Lorne J; Wozniak, Richard W

    2016-02-01

    Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

  16. Pattern recognition receptor signaling in human dendritic cells is enhanced by ICOS ligand and modulated by the Crohn's disease ICOSLG risk allele.

    PubMed

    Hedl, Matija; Lahiri, Amit; Ning, Kaida; Cho, Judy H; Abraham, Clara

    2014-05-15

    Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune homeostasis and cytokine secretion. Multiple loci are associated with IBD, but a functional explanation is missing for most. Here we found that pattern-recognition receptor (PRR)-induced cytokine secretion was diminished in human monocyte-derived dendritic cells (MDDC) from rs7282490 ICOSLG GG risk carriers. Homotypic interactions between the costimulatory molecule ICOS and the ICOS ligand on MDDCs amplified nucleotide-binding oligomerization domain 2 (NOD2)-initiated cytokine secretion. This amplification required arginine residues in the ICOSL cytoplasmic tail that recruited the adaptor protein RACK1 and the kinases PKC and JNK leading to PKC, MAPK, and NF-κB activation. MDDC from rs7282490 GG risk-carriers had reduced ICOSL expression and PRR-initiated signaling and this loss-of-function ICOSLG risk allele associated with an ileal Crohn's disease phenotype, similar to polymorphisms in NOD2. Taken together, ICOSL amplifies PRR-initiated outcomes, which might contribute to immune homeostasis.

  17. Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

    PubMed

    Tripathi, Jaindra N; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-08-01

    Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.

  18. Transgenic expression of the rice Xa21 pattern recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

    PubMed Central

    Tripathi, Jaindra Nath; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-01-01

    Summary Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, bio-control agents or resistant cultivars available to control BXW. Here we take advantage of the robust resistance conferred by the rice pattern recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21 mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic plants in the glass house for resistance against Xcm. About fifty percent of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the non-transgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa. PMID:24612254

  19. Calix[4]pyrrole derivative: recognition of fluoride and mercury ions and extracting properties of the receptor-based new material.

    PubMed

    de Namor, Angela F Danil; Khalife, Rasha

    2008-12-11

    A calix[4]pyrrole derivative, namely, meso-tetramethyl tetrakis (4-phenoxy methyl ketone) calix[4]pyrrole, 1, was synthesized and structurally (1H NMR) and thermodynamically characterized. The complexing properties of this receptor with a wide variety of anions and cations in dipolar aprotic media (acetonitrile, propylene carbonate, and dimethyl sulfoxide) were investigated through 1H NMR and conductance studies. The former technique was used to assess whether or not complexation occurs and if so to identify the active sites of interaction of 1 with ions. The composition of the complexes was established by conductance measurements. It was found that in dipolar aprotic solvents, 1 interacts only with two polluting ions (fluoride and mercury). The complexation thermodynamics of 1 and these ions in these solvents is reported. The medium effect on the binding process involving the fluoride ion is discussed taking into account the solvation properties of reactants and the product. Complexes of moderate stability are found. Given that this is an important factor to consider for the recycling of the loaded material in extraction processes, 1 was treated with formaldehyde in basic medium leading to the production of a calix[4]pyrrole based material able to extract fluoride and mercury (II) ions from water. Thus the optimum conditions for the extraction of these ions from aqueous solutions were established. The material is easily recyclable using an organic acid. Final conclusions are given. PMID:19053691

  20. Dehydro-β-proline Containing α4β1 Integrin Antagonists: Stereochemical Recognition in Ligand–Receptor Interplay

    PubMed Central

    2015-01-01

    A novel class of dehydro-β-proline-containing peptidomimetics, designed to be effective as α4β1 integrin ligands, has been developed on the basis of the fundamental requirements for the interactions of these transmembrane receptors with bioactive ligands. Dehydro-β-proline ring has been synthesized through an original pathway, involving ring closing metathesis of a diallylamino derivative. The synthesized products showed to be effective and selective as α4β1 integrin antagonists and displayed IC50 values in the nanomolar range in cell adhesion inhibition assays and in VCAM-1-induced phosphorylation of extracellular-signal-regulated kinases. Significant activity was observed also toward the homologous integrin α4β7, while they did not display any activity toward selected members of β1, β2, and β3 families. A strong dependence on the stereochemistry of the heterocyclic central core could be observed. The great importance of α4β1 integrin in chronic inflammatory and autoimmune diseases suggests a possible exploitation of these ligands as lead compounds for therapeutic tools development. PMID:26101577

  1. Structural Basis for Recognition of the Pore-Forming Toxin Intermedilysin by Human Complement Receptor CD59

    PubMed Central

    Johnson, Steven; Brooks, Nicholas J.; Smith, Richard A.G.; Lea, Susan M.; Bubeck, Doryen

    2013-01-01

    Summary Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here, we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this host-pathogen interaction. An ILY-derived peptide based on the binding site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway. PMID:23665225

  2. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.

    PubMed

    Karikó, Katalin; Buckstein, Michael; Ni, Houping; Weissman, Drew

    2005-08-01

    DNA and RNA stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). DNA containing methylated CpG motifs, however, is not stimulatory. Selected nucleosides in naturally occurring RNA are also methylated or otherwise modified, but the immunomodulatory effects of these alterations remain untested. We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. We conclude that nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue. PMID:16111635

  3. Calix[4]pyrrole derivative: recognition of fluoride and mercury ions and extracting properties of the receptor-based new material.

    PubMed

    de Namor, Angela F Danil; Khalife, Rasha

    2008-12-11

    A calix[4]pyrrole derivative, namely, meso-tetramethyl tetrakis (4-phenoxy methyl ketone) calix[4]pyrrole, 1, was synthesized and structurally (1H NMR) and thermodynamically characterized. The complexing properties of this receptor with a wide variety of anions and cations in dipolar aprotic media (acetonitrile, propylene carbonate, and dimethyl sulfoxide) were investigated through 1H NMR and conductance studies. The former technique was used to assess whether or not complexation occurs and if so to identify the active sites of interaction of 1 with ions. The composition of the complexes was established by conductance measurements. It was found that in dipolar aprotic solvents, 1 interacts only with two polluting ions (fluoride and mercury). The complexation thermodynamics of 1 and these ions in these solvents is reported. The medium effect on the binding process involving the fluoride ion is discussed taking into account the solvation properties of reactants and the product. Complexes of moderate stability are found. Given that this is an important factor to consider for the recycling of the loaded material in extraction processes, 1 was treated with formaldehyde in basic medium leading to the production of a calix[4]pyrrole based material able to extract fluoride and mercury (II) ions from water. Thus the optimum conditions for the extraction of these ions from aqueous solutions were established. The material is easily recyclable using an organic acid. Final conclusions are given.

  4. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  5. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor

    PubMed Central

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A.; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  6. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  7. Mutational analysis identifies leucine-rich repeat insertions crucial for pigeon toll-like receptor 7 recognition and signaling.

    PubMed

    Xiong, Dan; Song, Li; Jiao, Yang; Kang, Xilong; Chen, Xiang; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2015-11-15

    Toll-like receptor 7 (TLR7) is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, leading to the activation of the innate immune response. In this study, mutated pigeon TLR7 fragments, in which the insertion at position 10 of leucine-rich repeat 10 (LRR10) or at position 15 of LRR2/11/13/14 was deleted, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the Del10IN10 and Del14IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-α and TNF-α by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted. PMID:26553562

  8. Mutational analysis identifies leucine-rich repeat insertions crucial for pigeon toll-like receptor 7 recognition and signaling.

    PubMed

    Xiong, Dan; Song, Li; Jiao, Yang; Kang, Xilong; Chen, Xiang; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2015-11-15

    Toll-like receptor 7 (TLR7) is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, leading to the activation of the innate immune response. In this study, mutated pigeon TLR7 fragments, in which the insertion at position 10 of leucine-rich repeat 10 (LRR10) or at position 15 of LRR2/11/13/14 was deleted, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the Del10IN10 and Del14IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-α and TNF-α by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted.

  9. Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host.

    PubMed

    Núñez, José I; Molina, Nicolas; Baranowski, Eric; Domingo, Esteban; Clark, Stuart; Burman, Alison; Berryman, Stephen; Jackson, Terry; Sobrino, Francisco

    2007-08-01

    We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I(248)-->T in 2C, Q(44)-->R in 3A, and L(147)-->P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L(147)-->P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L(147)-->P, and this infection was inhibited by antibodies to alphavbeta6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin alphavbeta6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T(248)-->N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species. PMID:17522230

  10. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  11. Fluorescence switch on-off-on receptor constructed of quinoline allied calix[4]arene for selective recognition of Cu2+ from blood serum and F- from industrial waste water.

    PubMed

    Sutariya, Pinkesh G; Pandya, Alok; Lodha, Anand; Menon, Shobhana K

    2013-05-01

    A novel PET with ICT based substituted calix[4]arene fluroionophore was synthesized and used for detection of Cu(2+) and F(-) by emission spectra. The detection limit of the synthesized receptor was found to be 4.16 nM for Cu(2+) and 2.15 nM for F(-). Moreover, this probe has been applied for recognition of Cu(2+) in blood serum and F(-) from waste water.

  12. Exchange of the H(CC) domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin.

    PubMed

    Rummel, Andreas; Mahrhold, Stefan; Bigalke, Hans; Binz, Thomas

    2011-12-01

    The four-domain structure of botulinum neurotoxins (BoNTs) reflects their multistep intoxication process. The high toxicity of BoNTs primarily results from specific binding and uptake into neurons mediated by their 50-kDa cell-binding fragment (H(C) ). X-ray crystallography data have revealed that the H(C) fragment consists of two domains of equal size, named the 25-kDa N-terminal half of H(C) (H(CN) ) and the 25-kDa C-terminal half of H(C) (H(CC) ). In recent years, the ganglioside-binding sites of all seven BoNT serotypes have been allocated to the H(CC) domain. For BoNT/A, BoNT/B and BoNT/G, the protein receptor-binding site has been also been localized to the H(CC) domain. Here, we demonstrate that the H(CC) serotype can modulate the affinity of the H(C) fragment for neuronal membranes as well as the potency of full-length BoNT by replacing the BoNT/A H(CC) domain with the BoNT/B H(CC) , BoNT/C H(CC) and BoNT/E H(CC) domains, which exhibit higher affinity for synaptosomes. Indeed, the hybrids H(C) AB and H(C) AC display a higher affinity than wild-type H(C) A. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the H(CC) B domain (AAAB; letters represent the serotype origin of the four domains) was quadrupled as compared with wild-type BoNT/A. Analogously, exchange of the H(C) fragment (AABB) yielded a neurotoxin with four-fold higher potency. As BoNT/A and BoNT/B are extensively used to treat neurological disorders, thereby facing the problem of BoNT neutralizing antibody formation, a BoNT with increased potency would lower the repeatedly administered protein dosage while maintaining the clinical benefit. Such a lowered protein load will delay the onset of neurotoxin antibody formation in patients.

  13. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

    PubMed

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation. PMID:26185983

  14. Decreased Pattern Recognition Receptor Signaling, Interferon-Signature, and Bactericidal/Permeability-Increasing Protein Gene Expression in Cord Blood of Term Low Birth Weight Human Newborns

    PubMed Central

    Singh, Vikas Vikram; Chauhan, Sudhir Kumar; Rai, Richa; Kumar, Ashok; Singh, Shiva M.; Rai, Geeta

    2013-01-01

    Background Morbidity and mortality rates of low birth weight (LBW) newborns at term are higher than rates in normal birth weight (NBW) newborns. LBW newborns are at greater risk to acquire recurrent bacterial and viral infections during their first few weeks of life possibly as an outcome of compromised innate immune functions. As adaptive immunity is in a naive state, increased risk of infection of LBW as compared to NBW newborns may reflect impairments in innate immunity. Methodology To characterize the increased susceptibility to infections in LBW newborns we used microarray technology to identify differences in gene expression in LBW newborns (n = 8) compared to NBW newborns (n = 4) using cord blood. The results obtained from the microarray study were validated on a larger number of samples using real time RT-PCR (LBW = 22, NBW = 18) and western blotting (LBW = 12, NBW = 12). The Interferome database was used to identify interferon (IFN) signature genes and ingenuity pathway analysis identified canonical pathways and biological functions associated with the differentially expressed genes in LBW newborns. ELISAs for IFNs and bactericidal/permeability-increasing protein were performed in both LBW and NBW newborns and in adults (LBW = 18, NBW = 18, Adults  = 8). Principal Findings Upon microarray analysis, we identified 1,391 differentially expressed genes, of which, 1,065 genes were down-regulated and 326 genes were up-regulated in the LBW compared to NBW newborns. Of note, 70 IFN-signature genes were found to be significantly down-regulated in LBW compared to NBW newborns. Ingenuity pathway analysis revealed pattern recognition receptors signaling including Toll-Like Receptors (TLRs) -1, -5, and -8 genes and IFN signaling as the most significantly impacted pathways. Respiratory infectious diseases were the most significantly affected bio-functions in LBW newborns. Conclusion and Significance Diminished PRRs, IFN-signature, and

  15. Benzimidazole derivatives. 3. 3D-QSAR/CoMFA model and computational simulation for the recognition of 5-HT(4) receptor antagonists.

    PubMed

    López-Rodríguez, María L; Murcia, Marta; Benhamú, Bellinda; Viso, Alma; Campillo, Mercedes; Pardo, Leonardo

    2002-10-24

    A three-dimensional quantitative structure-affinity relationship study (3D-QSAR), using the comparative molecular field analysis (CoMFA) method, and subsequent computational simulation of ligand recognition have been successfully applied to explain the binding affinities for the 5-HT(4) receptor (5-HT(4)R) of a series of benzimidazole-4-carboxamides and carboxylates derivatives 1-24. The K(i) values of these compounds are in the range from 0.11 to 10 000 nM. The derived 3D-QSAR model shows high predictive ability (q(2) = 0.789 and r(2) = 0.997). Steric (contribution of 43.5%) and electrostatic (50.3%) fields and solvation energy (6.1%) of this novel class of 5-HT(4)R antagonists are relevant descriptors for structure-activity relationships. Computational simulation of the complexes between the benzimidazole-4-carboxamide UCM-21195 (5) and the carboxylate UCM-26995 (21) and a 3D model of the transmembrane domain of the 5-HT(4)R, constructed using the reported crystal structure of rhodopsin, have allowed us to define the molecular details of the ligand-receptor interaction that includes (i) the ionic interaction between the NH group of the protonated piperidine of the ligand and the carboxylate group of Asp(3.32), (ii) the hydrogen bond between the carbonyl oxygen of the ligand and the hydroxyl group of Ser(5.43), (iii) the hydrogen bond between the NH group of Asn(6.55) and the aromatic ring of carboxamides or the ether oxygen of carboxylates, (iv) the interaction of the electron-rich clouds of the aromatic ring of Phe(6.51) and the electron-poor hydrogens of the carbon atoms adjacent to the protonated piperidine nitrogen of the ligand, and (v) the pi-sigma stacking interaction between the benzimidazole system of the ligand and the benzene ring of Tyr(5.38). Moreover, the noticeable increase in potency at the 5-HT(4)R sites, by the introduction of a chloro or bromo atom at the 6-position of the aromatic ring, is attributed to the additional electrostatic and van der

  16. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

    PubMed Central

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M.

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  17. Molecular modeling of a T-cell receptor bound to a major histocompatibility complex molecule: implications for T-cell recognition.

    PubMed Central

    Almagro, J. C.; Vargas-Madrazo, E.; Lara-Ochoa, F.; Horjales, E.

    1995-01-01

    The main functions of the T-cell receptor (TCR) involve its specific interaction with short and linear antigenic peptides bound to the major histocompatibility complex (MHC) molecules. In the absence of a 3D structure for TCR and for the TCR/peptide/MHC complex, several attempts to characterize the structural components of the TCR/peptide/MHC interaction have been made. However, this subject is still troublesome. In this paper a computer-based 3D model for a TCR/peptide/MHC complex (5C.C7/moth cytochrome c [MCC] peptide 93-103/I-Ek) was obtained. The complex surface shows a high complementarity between the 5C.C7 structure and the peptide/I-Ek molecule. The mapping of residues involved in the TCR/peptide/MHC interaction shows close agreement with mutational experiments (Jorgensen JL, Reay PA, Ehrich EW, Davis MM, 1992b, Annu Rev Immunol 10:835-873). Moreover, the results are consistent with a recent variability analysis of TCR sequences using three variability indexes (Almagro JC, Zenteno-Cuevas R, Vargas-Madrazo E, Lara-Ochoa F, 1995b, Int J Pept Protein Res 45:180-186). Accordingly, the 3D model of the 5C.C7/MCC peptide 93-103/I-Ek complex provides a framework to generate testable hypotheses about TCR recognition. Thus, starting from this model, the role played by each loop that forms the peptide/MHC binding site of the TCR is discussed. PMID:8528069

  18. A novel junctional adhesion molecule A (CgJAM-A-L) from oyster (Crassostrea gigas) functions as pattern recognition receptor and opsonin.

    PubMed

    Liu, Conghui; Wang, Mengqiang; Jiang, Shuai; Wang, Lingling; Chen, Hao; Liu, Zhaoqun; Qiu, Limei; Song, Linsheng

    2016-02-01

    Junctional adhesion molecule (JAM), a subfamily of immunoglobulin superfamily (IgSF) with a couple of immunoglobulin domains, can act as regulator in homeostasis and inflammation of vertebrates. In the present study, a structural homolog of JAM-A (designated CgJAM-A-L) was screened out from oyster, Crassostrea gigas, through a search of JAM-A D1 domain (N-terminal Ig domain in JAM-A). The cDNA of CgJAM-A-L was of 1188 bp encoding a predicted polypeptide of 395 amino acids. The immunoreactive area of CgJAM-A-L mainly distributed over the plasma membrane of hemocytes. After Vibro splendidus or tumor necrosis factor (CgTNF-1) stimulation, the mRNA transcripts of CgJAM-A-L in hemocytes increased significantly by 4.46-fold and 9.00-fold (p < 0.01) of those in control group, respectively. The recombinant CgJAM-A-L protein (rCgJAM-A-L) could bind multiple PAMPs including lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), mannose (MAN), β-glucan (GLU) and poly(I:C), and various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibro anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L, and even increased more significantly after the pre-incubation of rCgJAM-A-L with microbes (p < 0.01). The results collectively indicated that CgJAM-A-L functioned as an important pattern recognition receptor (PRR) and opsonin in the immune defense against invading pathogen in oyster. Moreover, as the most primitive specie with homolog of JAMs, the information of CgJAM-A-L in oyster would provide useful clues for the evolutionary study of JAMs and immunoglobulins.

  19. Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif Is Required for Recognition by Toll-Like Receptor 11

    SciTech Connect

    K Kucera; A Koblansky; L Saunders; K Frederick; E De La Cruz; S Ghosh; Y Modis

    2011-12-31

    Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa such as Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor (TLR) 11 generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long {beta}-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the interleukin (IL)-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the {beta}-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and {beta}-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite Cryptosporidium parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from Plasmodium falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the {beta}-hairpin and diverges widely from the actin-binding surfaces of vertebrate profilins.

  20. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

    PubMed

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  1. Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I.

    PubMed

    Devarkar, Swapnil C; Wang, Chen; Miller, Matthew T; Ramanathan, Anand; Jiang, Fuguo; Khan, Abdul G; Patel, Smita S; Marcotrigiano, Joseph

    2016-01-19

    RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution. PMID:26733676

  2. Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: Sequential resonance assignment and implications for protein dynamics and receptor recognition

    SciTech Connect

    Hua, Qingxin ); Weiss, M.A. Massachusetts General Hospital, Boston, MA )

    1991-06-04

    The solution structure and dynamics of human insulin are ivestigated by 2D {sup 1}H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide (B26-B30) insulin. This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three {alpha}-helices and B-chain {beta}-turn) is similar to that observed in the 2-Zn crustal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structues. (3) residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To their knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening.

  3. Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

    PubMed Central

    Devarkar, Swapnil C.; Wang, Chen; Miller, Matthew T.; Ramanathan, Anand; Jiang, Fuguo; Khan, Abdul G.; Patel, Smita S.; Marcotrigiano, Joseph

    2016-01-01

    RNAs with 5′-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5′ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2′-O-methyl (2′-OMe) group to the 5′-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2′-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5′ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I’s ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5′ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5′OH, 5′ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2′-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2′-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution. PMID:26733676

  4. Molecular basis of non-self recognition by the horseshoe crab tachylectins.

    PubMed

    Kawabata, Shun-ichiro; Tsuda, Ryoko

    2002-09-19

    The self/non-self discrimination by innate immunity through simple ligands universally expressed both on pathogens and hosts, such as monosaccharides and acetyl group, depends on the density or clustering patterns of the ligands. The specific recognition by the horseshoe crab tachylectins with a propeller-like fold or a propeller-like oligomeric arrangement is reinforced by the short distance between the individual binding sites that interact with pathogen-associated molecular patterns (PAMPs). There is virtually no conformational change in the main or side chains of tachylectins upon binding with the ligands. This low structural flexibility of the propeller structures must be very important for specific interaction with PAMPs. Mammalian lectins, such as mannose-binding lectin and ficolins, trigger complement activation through the lectin pathway in the form of opsonins. However, tachylectins have no effector collagenous domains and no lectin-associated serine proteases found in the mammalian lectins. Furthermore, no complement-like proteins have been found in horseshoe crabs, except for alpha(2)-macroglobulin. The mystery of the molecular mechanism of the scavenging pathway of pathogens in horseshoe crabs remains to be solved.

  5. The low-density lipoprotein receptor-related protein 1 and amyloid-β clearance in Alzheimer’s disease

    PubMed Central

    Kanekiyo, Takahisa; Bu, Guojun

    2014-01-01

    Accumulation and aggregation of amyloid-β (Aβ) peptides in the brain trigger the development of progressive neurodegeneration and dementia associated with Alzheimer’s disease (AD). Perturbation in Aβ clearance, rather than Aβ production, is likely the cause of sporadic, late-onset AD, which accounts for the majority of AD cases. Since cellular uptake and subsequent degradation constitute a major Aβ clearance pathway, the receptor-mediated endocytosis of Aβ has been intensely investigated. Among Aβ receptors, the low-density lipoprotein receptor-related protein 1 (LRP1) is one of the most studied receptors. LRP1 is a large endocytic receptor for more than 40 ligands, including apolipoprotein E, α2-macroglobulin and Aβ. Emerging in vitro and in vivo evidence demonstrates that LRP1 is critically involved in brain Aβ clearance. LRP1 is highly expressed in a variety of cell types in the brain including neurons, vascular cells and glial cells, where LRP1 functions to maintain brain homeostasis and control Aβ metabolism. LRP1-mediated endocytosis regulates cellular Aβ uptake by binding to Aβ either directly or indirectly through its co-receptors or ligands. Furthermore, LRP1 regulates several signaling pathways, which also likely influences Aβ endocytic pathways. In this review, we discuss how LRP1 regulates the brain Aβ clearance and how this unique endocytic receptor participates in AD pathogenesis. Understanding of the mechanisms underlying LRP1-mediated Aβ clearance should enable the rational design of novel diagnostic and therapeutic strategies for AD. PMID:24904407

  6. Galantamine ameliorates the impairment of recognition memory in mice repeatedly treated with methamphetamine: involvement of allosteric potentiation of nicotinic acetylcholine receptors and dopaminergic-ERK1/2 systems.

    PubMed

    Noda, Yukihiro; Mouri, Akihiro; Ando, Yu; Waki, Yukari; Yamada, Shin-Nosuke; Yoshimi, Akira; Yamada, Kiyofumi; Ozaki, Norio; Wang, Dayong; Nabeshima, Toshitaka

    2010-11-01

    Galantamine, a drug used to treat Alzheimer's disease, inhibits acetylcholinesterase (AChE) and allosterically modulates nicotinic acetylcholine receptors (nAChRs) resulting in stimulation of catecholamine neurotransmission. In this study, we investigated whether galantamine exerts cognitive-improving effects through the allosteric modulation of nAChRs in an animal model of methamphetamine (Meth) psychosis. The mice treated with Meth (1 mg/kg.d) for 7 d showed memory impairment in a novel object recognition test. Galantamine (3 mg/kg) ameliorated the memory impairment, and it increased the extracellular dopamine release in the prefrontal cortex (PFC) of Meth-treated mice. Donepezil, an AChE inhibitor (1 mg/kg) increased the extracellular ACh release in the PFC, whereas it had no effect on the memory impairment in Meth-treated mice. The nAChR antagonist, mecamylamine, and dopamine D1 receptor antagonist, SCH 23390, blocked the ameliorating effect of galantamine on Meth-induced memory impairment, whereas the muscarinic AChR antagonist, scopolamine, had no effect. The effects of galantamine on extracellular dopamine release were also antagonized by mecamylamine. Galantamine attenuated the defect of the novelty-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). The ameliorating effect of galantamine on recognition memory in Meth-treated mice was negated by microinjection of an ERK inhibitor, PD98059, into the PFC. These results suggest that the ameliorating effect of galantamine on Meth-induced memory impairment is associated with indirect activation of dopamine D1 receptor-ERK1/2 following augmentation with dopaminergic neurotransmission in the PFC through the allosteric activation of nAChRs. Galantamine could be a useful therapeutic agent for treating cognitive deficits in schizophrenia/Meth psychosis, as well as Alzheimer's disease.

  7. Arginine vasopressin 1a receptor RS3 promoter microsatellites in schizophrenia: a study of the effect of the "risk" allele on clinical symptoms and facial affect recognition.

    PubMed

    Golimbet, Vera; Alfimova, Margarita; Abramova, Lilia; Kaleda, Vasily; Gritsenko, Inga

    2015-02-28

    We studied AVPR1A RS3 polymorphism in schizophrenic patients and controls. AVPR1A RS3 was not associated with schizophrenia. The allele 327bp implicated in autism and social behavior was associated with negative symptoms and tended to be linked to patient facial affect recognition suggesting its impact on schizophrenia social phenotypes.

  8. Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging.

    PubMed

    Chtcheglova, Lilia A; Atalar, Fatmahan; Ozbek, Ugur; Wildling, Linda; Ebner, Andreas; Hinterdorfer, Peter

    2008-04-01

    The inhibition of the human ether-à-go-go-related (hERG) K+ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias. Ergtoxin 1 (ErgTx1) belongs to scorpion-toxins, which are K+ channel-blockers, and binds to hERG channel with 1:1 stoichiometry and high affinity (Kd approximately 10 nM). Nevertheless, patch-clamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents, even at high ErgTx1 concentrations. Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG. In this study, simultaneous topography and recognition imaging (TREC) on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel (on S1-S2 region) for Anti-Kv11.1 (hERG-extracellular-antibody). The recognition maps of hERG channels contained recognition spots, haphazardly distributed and organized in clusters. Recognition images after the addition of ErgTx1 at high concentrations ( approximately 1 microM) revealed subsequent partial disappearance of clusters, indicating that ErgTx1 was bound to the S1-S2 region. These results were supported by AFM force spectroscopy data, showing for the first time that voltage sensing domain (S1-S4) of hERG K+ channel might be one of the multiple binding sites of ErgTx1. PMID:18286302

  9. Novel salicylic acid-oriented thiourea-type receptors as colorimetric chemosensor: Synthesis, characterizations and selective naked-eye recognition properties

    NASA Astrophysics Data System (ADS)

    Li, Shaowei; Cao, Xiufang; Chen, Changshui; Ke, Shaoyong

    2012-10-01

    Based on the salicylic acid backbone, three highly sensitive and selective colorimetric chemosensors with an acylthiourea binding unit have been designed, synthesized and characterized. These chemosensors have been utilized for selective recognition of fluoride anions in dry DMSO solution by typical spectroscopic titration techniques. Furthermore, the obtained chemosensors AR1-3 have shown naked-eye sensitivity for detection of biologically important fluoride ion over other anions in solution.

  10. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor.

  11. Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands*

    PubMed Central

    Sergeev, Eugenia; Hansen, Anders Højgaard; Pandey, Sunil K.; MacKenzie, Amanda E.; Hudson, Brian D.; Ulven, Trond; Milligan, Graeme

    2016-01-01

    Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor. PMID:26518871

  12. The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11-Pro16 beta-hairpin region in the recognition mechanism.

    PubMed

    Destoumieux-Garzón, Delphine; Duquesne, Sophie; Peduzzi, Jean; Goulard, Christophe; Desmadril, Michel; Letellier, Lucienne; Rebuffat, Sylvie; Boulanger, Pascale

    2005-08-01

    The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

  13. Combined serotonin (5-HT)1A agonism, 5-HT(2A) and dopamine D₂ receptor antagonism reproduces atypical antipsychotic drug effects on phencyclidine-impaired novel object recognition in rats.

    PubMed

    Oyamada, Yoshihiro; Horiguchi, Masakuni; Rajagopal, Lakshmi; Miyauchi, Masanori; Meltzer, Herbert Y

    2015-05-15

    Subchronic administration of an N-methyl-D-aspartate receptor (NMDAR) antagonist, e.g. phencyclidine (PCP), produces prolonged impairment of novel object recognition (NOR), suggesting they constitute a hypoglutamate-based model of cognitive impairment in schizophrenia (CIS). Acute administration of atypical, e.g. lurasidone, but not typical antipsychotic drugs (APDs), e.g. haloperidol, are able to restore NOR following PCP (acute reversal model). Furthermore, atypical APDs, when co-administered with PCP, have been shown to prevent development of NOR deficits (prevention model). Most atypical, but not typical APDs, are more potent 5-HT(2A) receptor inverse agonists than dopamine (DA) D2 antagonists, and have been shown to enhance cortical and hippocampal efflux and to be direct or indirect 5-HT(1A) agonists in vivo. To further clarify the importance of these actions to the restoration of NOR by atypical APDs, sub-effective or non-effective doses of combinations of the 5-HT(1A) partial agonist (tandospirone), the 5-HT(2A) inverse agonist (pimavanserin), or the D2 antagonist (haloperidol), as well as the combination of all three agents, were studied in the acute reversal and prevention PCP models of CIS. Only the combination of all three agents restored NOR and prevented the development of PCP-induced deficit. Thus, this triple combination of 5-HT(1A) agonism, 5-HT(2A) antagonism/inverse agonism, and D2 antagonism is able to mimic the ability of atypical APDs to prevent or ameliorate the PCP-induced NOR deficit, possibly by stimulating signaling cascades from D1 and 5-HT(1A) receptor stimulation, modulated by D2 and 5-HT(2A) receptor antagonism. PMID:25448429

  14. Molecular recognition of metabotropic glutamate receptor type 1 (mGluR1): synergistic understanding with free energy perturbation and linear response modeling.

    PubMed

    Kang, Seung-gu; Das, Payel; McGrane, Scott J; Martin, Alan J; Huynh, Tien; Royyuru, Ajay K; Taylor, Andrew J; Jones, Paul G; Zhou, Ruhong

    2014-06-19

    Metabotropic glutamate receptors (mGluRs) constitute an important family of the G-protein coupled receptors. Due to their widespread distribution in the central nervous system (CNS), these receptors are attractive candidates for understanding the molecular basis of various cognitive processes as well as for designing inhibitors for relevant psychiatric and neurological disorders. Despite many studies on drugs targeting the mGluR receptors to date, the molecular level details on the ligand binding dynamics still remain unclear. In this study, we performed in silico experiments for mGluR1 with 29 different ligands including known synthetic agonists and antagonists as well as natural amino acids. The ligand-receptor binding affinities were estimated by the use of atomistic simulations combined with the mathematically rigorous, Free Energy Perturbation (FEP) method, which successfully recognized the native agonist l-glutamate among the highly favorable binders, and also accurately distinguished antagonists from agonists. Comparative contact analysis also revealed the binding mode differences between natural and non-natural amino acid-based ligands. Several factors potentially affecting the ligand binding affinity and specificity were identified including net charges, dipole moments, and the presence of aromatic rings. On the basis of these findings, linear response models (LRMs) were built for different sets of ligands that showed high correlations (R(2) > 0.95) to the corresponding FEP binding affinities. These results identify some key factors that determine ligand-mGluR1 binding and could be used for future inhibitor designs and support a role for in silico modeling for understanding receptor ligand interactions.

  15. Identification of a crucial energetic footprint on the alpha1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors.

    PubMed

    Baker, B M; Turner, R V; Gagnon, S J; Wiley, D C; Biddison, W E

    2001-03-01

    Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule. PMID:11238586

  16. Molecular Basis for the Recognition of Structurally Distinct Autoinducer Mimics by the Pseudomonas aeruginosa LasR Quorum-Sensing Signaling Receptor

    SciTech Connect

    Zou, Yaozhong; Nair, Satish K.

    2010-01-12

    The human pathogen Pseudomonas aeruginosa coordinates the expression of virulence factors using quorum sensing, a signaling cascade triggered by the activation of signal receptors by small-molecule autoinducers. These homoserine lactone autoinducers stabilize their cognate receptors and activate their functions as transcription factors. Because quorum sensing regulates the progression of infection and host immune resistance, significant efforts have been devoted toward the identification of small molecules that disrupt this process. Screening efforts have identified a class of triphenyl compounds that are structurally distinct from the homoserine lactone autoinducer, yet interact specifically and potently with LasR receptor to modulate quorum sensing (Muh et al., 2006a). Here we present the high-resolution crystal structures of the ligand binding domain of LasR in complex with the autoinducer N-3-oxo-dodecanoyl homoserine lactone (1.4 {angstrom} resolution), and with the triphenyl mimics TP-1, TP-3, and TP-4 (to between 1.8 {angstrom} and 2.3 {angstrom} resolution). These crystal structures provide a molecular rationale for understanding how chemically distinct compounds can be accommodated by a highly selective receptor, and provide the framework for the development of novel quorum-sensing regulators, utilizing the triphenyl scaffold.

  17. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  18. Characterization of Murine Cytomegalovirus m157 from Infected Cells and Identification of Critical Residues Mediating Recognition by the NK Cell Receptor, Ly49H

    PubMed Central

    Davis, Aja H.; Guseva, Natalya V.; Ball, Brianne L.; Heusel, Jonathan W.

    2008-01-01

    Activated natural killer (NK) cells mediate potent cytolytic and secretory effector functions, and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize major histocompatibility class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the m157 glycoprotein of murine cytomegalovirus (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) in C57BL/6 mice, and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157 we identify unique residues that provide for efficient cell surface expression of m157, but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical α0-helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand. PMID:18566392

  19. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2.

    PubMed

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I B; Schornack, Sebastian; Jones, Alexandra M E; Bozkurt, Tolga O; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  20. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

    PubMed Central

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I. B.; Schornack, Sebastian; Jones, Alexandra M. E.; Bozkurt, Tolga O.; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  1. A retrievable, water-soluble and biocompatible fluorescent probe for recognition of Cu(II) and sulfide based on a peptide receptor.

    PubMed

    Hao, Yuanqiang; Chen, Wansong; Wang, Liqiang; Zhu, Xu; Zhang, Yintang; Qu, Peng; Liu, Lin; Zhou, Binbin; Liu, You-Nian; Xu, Maotian

    2015-10-01

    A novel fluorescent probe C-GGH for recognition of Cu(2+) and S(2-) was designed and synthesized by incorporation a natural tripeptide GGH (Gly-Gly-His) moiety, which acts as a recognition unit for Cu(2+), to a coumarin fluorophore. C-GGH displayed ON-OFF type fluorescence response towards Cu(2+) with high selectivity in 100% aqueous solution. Furthermore, the in situ generated non-fluorescent C-GGH-Cu(2+) complex can serve as an effective OFF-ON type fluorescent probe for selective sensing sulfide anion. Due to the strong binding affinity of S(2-) to Cu(2+), sulfide anion can extract Cu(2+) from C-GGH-Cu(2+) complex, resulting the release of C-GGH and the revival of fluorescent emission of the system. Techniques of emission spectrometry, NMR, mass spectrometry, and HPLC were used to confirm the displacement mechanism of C-GGH-Cu(2+) for sensing sulfide anion. The probe C-GGH-Cu(2+) allowed detection of S(2-) in aqueous buffer solution with a LOD (limit of detection) of 0.44μM. By taking advantages of the natural tripeptide GGH, the sensor exhibits excellent biocompatibility and water solubility. Moreover, the cell imaging study demonstrated that the probe can be exploited to detect sulfur anion in biological systems. PMID:26078164

  2. A retrievable, water-soluble and biocompatible fluorescent probe for recognition of Cu(II) and sulfide based on a peptide receptor.

    PubMed

    Hao, Yuanqiang; Chen, Wansong; Wang, Liqiang; Zhu, Xu; Zhang, Yintang; Qu, Peng; Liu, Lin; Zhou, Binbin; Liu, You-Nian; Xu, Maotian

    2015-10-01

    A novel fluorescent probe C-GGH for recognition of Cu(2+) and S(2-) was designed and synthesized by incorporation a natural tripeptide GGH (Gly-Gly-His) moiety, which acts as a recognition unit for Cu(2+), to a coumarin fluorophore. C-GGH displayed ON-OFF type fluorescence response towards Cu(2+) with high selectivity in 100% aqueous solution. Furthermore, the in situ generated non-fluorescent C-GGH-Cu(2+) complex can serve as an effective OFF-ON type fluorescent probe for selective sensing sulfide anion. Due to the strong binding affinity of S(2-) to Cu(2+), sulfide anion can extract Cu(2+) from C-GGH-Cu(2+) complex, resulting the release of C-GGH and the revival of fluorescent emission of the system. Techniques of emission spectrometry, NMR, mass spectrometry, and HPLC were used to confirm the displacement mechanism of C-GGH-Cu(2+) for sensing sulfide anion. The probe C-GGH-Cu(2+) allowed detection of S(2-) in aqueous buffer solution with a LOD (limit of detection) of 0.44μM. By taking advantages of the natural tripeptide GGH, the sensor exhibits excellent biocompatibility and water solubility. Moreover, the cell imaging study demonstrated that the probe can be exploited to detect sulfur anion in biological systems.

  3. Design, synthesis and 1H NMR study of C3v-symmetric anion receptors with urethane-NH as recognition group

    NASA Astrophysics Data System (ADS)

    Park, Jin-Oh; Sahoo, Suban K.; Choi, Heung-Jin

    2016-01-01

    C3v-Symmetric anion receptors 3 and 4 with urethane groups were synthesized by using trindane triol as tripodal molecular framework. In 1H NMR titration study, the receptors showed noticeable downfield shift/disappearance of the urethane-NH peak in presence of H2PO4- and F- due to the host-guest complexation occurred through multiple hydrogen bonding and/or the deprotonation of urethane-NH groups. Other tested anions such as Cl-, Br-, HSO4-, and NO3- showed either no or negligible chemical shift of the urethane groups. The deprotonation event in 4 allowed selective detection of F- by perceptible color change from colorless to yellowish-red with the appearance of a new charge transfer absorption band at 450 nm.

  4. Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with alpha1beta2gamma2S subunits of GABA (A) receptors.

    PubMed

    Pericić, Danka; Strac, Dubravka Svob; Jembrek, Maja Jazvinsćak; Vlainić, Josipa

    2007-05-01

    Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABA(A) receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors were exposed for 72 h to a high concentration of diazepam (50 microM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam and [(3)H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B (max)) of [(3)H]flunitrazepam and [(3)H]muscimol binding sites in the membranes, and of [(3)H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K (d)) of binding sites. The diazepam-induced increase in the B (max) value of [(3)H]flunitrazepam binding sites was reduced by two GABA(A) receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). In addition, it was reduced by cycloheximide (5 microg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 microg/ml), an RNA synthesis inhibitor. Flumazenil (5 microM), the antagonist of benzodiazepine binding sites, also up-regulated [(3)H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from diazepam (50 microM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABA(A) receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [(3)H]muscimol binding sites

  5. Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.

    PubMed

    Ding, Sai; Zhang, Jing; Tian, Yu; Huang, Baolin; Yuan, Yuan; Liu, Changsheng

    2016-09-01

    Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration. PMID:27156155

  6. A single lysine of the two-lysine recognition motif of the D3 domain of receptor-associated protein is sufficient to mediate endocytosis by low-density lipoprotein receptor-related protein.

    PubMed

    van den Biggelaar, Maartje; Sellink, Erica; Klein Gebbinck, Jacqueline W T M; Mertens, Koen; Meijer, Alexander B

    2011-03-01

    Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by complement-type repeats (CR) each comprising a binding pocket for a single basic amino acid residue. It has been proposed that at least two CRs are required for high-affinity interaction by utilising two spatially distinct lysine residues on the ligand surface. LDL receptor-related protein (LRP) mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We now addressed the role of the two critical lysines not only in LRP binding but also in LRP-dependent endocytosis. Variants of the third domain (D3) of receptor-associated protein (RAP) were created carrying lysine to alanine or arginine replacements at the putative contact residues K253, K256 and K270. Surface plasmon resonance revealed that replacement of K253 did not affect high-affinity LRP binding at all, whereas replacement of either K256 or K270 markedly reduced the affinity by approximately 10-fold. Binding was abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding. This suggests that despite their positive charge, arginine residues do not support receptor binding at all. Confocal microscopy and flow cytometry studies surprisingly revealed that the single mutants were still taken up and still competed for the uptake of full length RAP despite their receptor binding defect. We therefore propose that the presence of only one of the two critical lysines is sufficient to drive endocytosis. PMID:21144910

  7. Dynamic chemistry of anion recognition

    SciTech Connect

    Custelcean, Radu

    2012-01-01

    In the past 40 years, anion recognition by synthetic receptors has grown into a rich and vibrant research topic, developing into a distinct branch of Supramolecular Chemistry. Traditional anion receptors comprise organic scaffolds functionalized with complementary binding groups that are assembled by multistep organic synthesis. Recently, a new approach to anion receptors has emerged, in which the host is dynamically self-assembled in the presence of the anionic guest, via reversible bond formation between functional building units. While coordination bonds were initially employed for the self-assembly of the anion hosts, more recent studies demonstrated that reversible covalent bonds can serve the same purpose. In both cases, due to their labile connections, the molecular constituents have the ability to assemble, dissociate, and recombine continuously, thereby creating a dynamic combinatorial library (DCL) of receptors. The anionic guests, through specific molecular recognition, may then amplify (express) the formation of a particular structure among all possible combinations (real or virtual) by shifting the equilibria involved towards the most optimal receptor. This approach is not limited to solution self-assembly, but is equally applicable to crystallization, where the fittest anion-binding crystal may be selected. Finally, the pros and cons of employing dynamic combinatorial chemistry (DCC) vs molecular design for developing anion receptors, and the implications of both approaches to selective anion separations, will be discussed.

  8. Tumor recognition and lytic competence of IL-2-activated lymphocytes: regulation of both antibody-independent and -dependent cellular cytotoxicity via P75 IL-2 receptor.

    PubMed

    Lagoo-Deenadayalan, S; Lagoo, A S; Hardy, K J; Grimm, E A

    1992-08-01

    Fc receptor-positive lymphocytes (FcR+) contain lymphokine-activated killer cell (LAK) precursors that in response to IL-2 develop potent antitumor cytotoxicity. These FcR+ cells are also capable of antibody-dependent cytotoxicity (ADCC), which can be detected using fresh human peripheral blood lymphocytes (PBL) directed to murine targets, however, PBL-mediated ADCC to human tumors usually is very low, requiring a stimulation of the PBL, which also can be accomplished with IL-2. Using human melanoma tumor target cells, with and without the 14G2a monoclonal antibody, we examined in parallel the role of p75 IL-2 receptor for regulation of the induction of both LAK and ADCC forms of antitumor cytotoxicity. Enrichment of FcR+ cells from fresh peripheral blood by elutriation and flow cytometry, followed by varying periods of IL-2 culture, revealed a differential kinetics of activation. ADCC was detectable after PBL exposure to IL-2 for as short as the 4 h cytotoxicity assay, while LAK activation required more than 24 h of exposure. Elimination of the FcR+ cells by magnetic bead depletion from large granular lymphocyte populations (LGL) resulted in a loss of both LAK and ADCC. Addition of ant