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Ligand Binding (ligand + binding)
Terms modified by Ligand Binding Selected AbstractsPyrene Excimer Fluorescence of Yeast Alcohol Dehydrogenase: A Sensitive Probe to Investigate Ligand Binding and Unfolding Pathway of the EnzymePHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2006Manas Kumar Santra ABSTRACT The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guani-dinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH. [source] Phosphorylated Human Lectin Galectin-3: Analysis of Ligand Binding by Histochemical Monitoring of Normal/Malignant Squamous Epithelia and by Isothermal Titration CalorimetryANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009P. Szabo Summary The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology. [source] Ligand binding of leukocyte integrin very late antigen-4 involves exposure of sulfhydryl groups and is subject to redox modulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008Si-Yen Liu Abstract Activation of leukocyte integrins is important for selective recruitment of cells from the circulation to tissues. Our previous studies showed that the binding between the integrin very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1) is modulated by reactive oxygen species. In this study, we investigated the molecular nature of redox modulation on the activation states of VLA-4 on human leukocytes. We found that ligand binding of VLA-4 induced exposure of sulfhydryl groups on the ,4 peptide. Low concentrations (5,10,µM) of exogenous hydrogen peroxide in the presence or absence of added glutathione enhanced the ligand binding ability of VLA-4 to VCAM-1 and cell rolling on VCAM-1, while higher concentrations (,100,µM) of hydrogen peroxide inhibited the binding. Exogenous hydrogen peroxide and glutathione induced molecular modification of S -glutathionylation on the ,4 peptide. The redox regulation of the VLA-4 binding activity required outside-in signaling and cytoskeleton rearrangement. Our results indicate that ligand binding of VLA-4 involves redox modulations which may play a pivotal role in regulating the activation states of VLA-4 in inflammatory tissues and hence direct leukocyte trafficking. [source] Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracleFEBS JOURNAL, Issue 13 2002Markus Lezzi The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. [source] Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamicsMASS SPECTROMETRY REVIEWS, Issue 1 2008Sven Schuchardt Abstract Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein,protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N -terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling. © 2007 Wiley Periodicals, Inc., Mass Spec Rev 27:51,65, 2008 [source] Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexesPROTEIN SCIENCE, Issue 4 2001Toshie Yoneyama BH4, 6R - l - erythro -5,6,7,8-tetrahydrobiopterin; GFRP, GTP cyclohydrolase I feedback regulatory protein Abstract GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R - l - erythro -5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 ,M, and phenylalanine binds to the protein complex with a Kd of 94 ,M. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively. [source] Ligand binding at the transthyretin dimer,dimer interface: structure of the transthyretin,T4Ac complex at 2.2,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2005Vivian Cody The crystal structure of the complex of human transthyretin (hTTR) with 3,3,,5,5,-tetraiodothyroacetic acid (T4Ac) has been determined to 2.2,Å resolution. The complex crystallizes in the orthorhombic space group P21212, with unit-cell parameters a = 43.46, b = 85.85, c = 65.44,Å. The structure was refined to R = 17.3% and Rfree = 21.9% for reflections without any ,-cutoff. T4Ac is bound in both the forward and the reverse mode in the two binding sites of hTTR. In the forward orientation, T4Ac binds in a position similar to that described for thyroxine (T4) in the orthorhombic hTTR,T4 complex. In this orientation, the iodine substituents of the phenolic ring are bound in the P3,/P2 halogen pockets. In the reverse orientation, which is the major binding mode of T4Ac, the ligand is bound deep in the TTR channel, with the carboxylic group bound in the P3, pocket and forming simultaneous polar interactions with the residues constituting the two hormone-binding sites. Such interactions of a thyroxine-analogue ligand bound in the reverse mode have never been observed in TTR complexes previously. [source] Phosphatidylinositol-3-OH kinase regulatory subunits are differentially expressed during development of the rat cerebellumDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2001José L. Trejo Abstract Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin-like growth factor I (IGF-I) most likely represents the main survival signal during neuronal differentiation. IGF-I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF-I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3-kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110, or ,) associated with one of a large family of regulatory subunits (p85,, p85,, p55,, p55,, and p50,). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55, regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55, is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF-IR. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 39,50, 2001 [source] Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresisELECTROPHORESIS, Issue 16 2008Ruth E. Montes Abstract This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box,Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [Lo], on protein,neutral ligand binding. Statistical analysis results were used to create a model for response surface prediction via contour and surface plots at a given maximum response (,RMTR) to reach a targeted Kb,=,2.50×106,M,1. The adequacy of the model was then validated by experimental runs at the optimal predicted solution (injection time,=,2.3,min, voltage,=,11.6,kV, [Lo],=,1.4,,M). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor,ligand combinations. [source] Implementation of chemometric methodology in ACE: Predictive investigation of protein,ligand bindingELECTROPHORESIS, Issue 16 2007Grady Hanrahan Abstract An ACE predictive investigation of protein,ligand binding using a highly effective chemometric response surface design technique is presented. Here, Kd was estimated using one noninteracting standard which relates to changes in the electrophoretic mobility of carbonic anhydrase B (CAB, EC 4.2.1.1) on complexation with the ligand 4-carboxybenzenesulfonamide (CBSA) present in the electrophoresis buffer. Experimental factors including injection time, capillary length, and applied voltage were selected and tested at three levels in a Box,Behnken design. Statistical analysis results were used to create a mathematical model for response surface prediction via contour and surface plots at a given target response (Kd,=,1.19×10,6,M). As expected, there were a number of predicted solutions that reached our target response based on the significance of each factor at appropriate levels. The adequacy of the model was validated by experimental runs with the predicted model solution (capillary length,=,47,cm, voltage,=,11,kV, injection time,=,0.01,min) presented in detail as an example. [source] Transcription factor Fli-1 expression by bone marrow cells in chronic myeloproliferative disorders is independent of an underlying JAK2 (V617F) mutationEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2006Oliver Bock Abstract:,Objectives:,Friend leukemia integration-1 (Fli-1), a member of the Ets gene family of transcription factors, has been demonstrated to be a target of a leukaemia inducing virus in mice, and is known to be part of a fusion gene in Ewings' sarcoma in humans. Wild-type Fli-1 is involved in lineage commitment of megakaryocytes and myeloid progenitors through induction of Janus kinases (JAKs) following ligand binding to cytokine and growth factor receptors. Proliferation of atypical megakaryocytes is a predominant histopathological feature in Philadelphia chromosome negative chronic myeloproliferative disorders (Ph, CMPD) and a potential aberrant expression of Fli-1 has not been investigated so far. Methods:,Fli-1 expression was investigated by real-time RT-PCR and immunohistochemistry in bone marrow cells derived from Ph, CMPD (n = 80) and non-neoplastic haematopoiesis (n = 21) following determination of the JAK2 status. Results:,Fli-1 mRNA expression was significantly higher in Essential thrombocythaemia (ET) with JAK2 (V617F) compared with other Ph, CMPD and control (P < 0.001). By immunohistochemistry, Fli-1 protein could be detected in nuclei of atypical megakaryocytes in Ph, CMPD and, less accentuated, in non-neoplastic megakaryocytes. Fli-1 protein expression by myeloid progenitors was considerably heterogenous in Ph, CMPD independent of an underlying JAK2 (V617F) mutation and without notable differences to non-neoplastic haematopoiesis. Conclusion:,Fli-1 is rather constitutively expressed by bone marrow cells in Ph, CMPD independent of the underlying JAK2 status. The overall stronger labelling for Fli-1 in megakaryocytes in Ph, CMPD most likely reflects the degree of polyploidisation but aberrant activation of nuclear target genes can not be excluded. [source] Site-directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2008Sarah Abstract Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N,terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6. [source] Osteopontin as a new player in mast cell biologyEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008Silvia Bulfone-Paus Abstract The secreted glycoprotein osteopontin (OPN) sets into motion an astounding variety of activities that range from bone remodeling via immunomodulation to the inhibition of apoptosis. In the current issue of the European Journal of Immunology, OPN now also enters mast cell biology and the regulation of IgE-dependent immune responses since it is reported that connective tissue-type mast cells from fetal murine skin constitutively secrete biologically active OPN. Moreover, it is shown that, in vitro, OPN augments IgE-mediated mast cell degranulation and migration via ligand binding to cognate OPN receptors on the mast cell surface (CD44, ,v integrin) and that the magnitude of an IgE-mediated passive cutaneous anaphylaxis reaction is augmented by OPN in vivo. Here, we discuss why this newly discovered property of OPN fits well into the emerging concept that OPN may serve as a multi-purpose environmental damage-response protein. See accompanying article: http://dx.doi.org/10.1002/eji200737057 [source] Ligand binding of leukocyte integrin very late antigen-4 involves exposure of sulfhydryl groups and is subject to redox modulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008Si-Yen Liu Abstract Activation of leukocyte integrins is important for selective recruitment of cells from the circulation to tissues. Our previous studies showed that the binding between the integrin very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1) is modulated by reactive oxygen species. In this study, we investigated the molecular nature of redox modulation on the activation states of VLA-4 on human leukocytes. We found that ligand binding of VLA-4 induced exposure of sulfhydryl groups on the ,4 peptide. Low concentrations (5,10,µM) of exogenous hydrogen peroxide in the presence or absence of added glutathione enhanced the ligand binding ability of VLA-4 to VCAM-1 and cell rolling on VCAM-1, while higher concentrations (,100,µM) of hydrogen peroxide inhibited the binding. Exogenous hydrogen peroxide and glutathione induced molecular modification of S -glutathionylation on the ,4 peptide. The redox regulation of the VLA-4 binding activity required outside-in signaling and cytoskeleton rearrangement. Our results indicate that ligand binding of VLA-4 involves redox modulations which may play a pivotal role in regulating the activation states of VLA-4 in inflammatory tissues and hence direct leukocyte trafficking. [source] Comparison of the inward- and outward-open homology models and ligand binding of human P-glycoproteinFEBS JOURNAL, Issue 23 2009Ilza K. Pajeva An homology model of human P-glycoprotein, based on the X-ray structure of the recently resolved mouse P-glycoprotein, is presented. The model corresponds to the inward-facing conformation competent for drug binding. From the model, the residues involved in the protein-binding cavity are identified and compared with those in the outward-facing conformation of human P-glycoprotein developed previously based on the Sav1866 structure. A detailed analysis of the interactions of the cyclic peptides QZ59- RRR and QZ59- SSS is presented in both the X-ray structures of mouse P-glycoprotein and the human P-glycoprotein model generated by ligand docking. The results confirm the functional role of transmembrane domains TM4, TM6, TM10 and TM12 as entrance gates to the protein cavity, and also imply differences in their functions. The analysis of the cavities in both models suggests that the ligands remain bound to the same residues during the transition from the inward- to the outward-facing conformations. The analysis of the ligand,protein interactions in the X-ray complexes shows differences in the residues involved, as well as in the specific interactions performed by the same ligand within the same protein. This observation is supported by docking of the QZ59 ligands into human P-glycoprotein, thus aiding in the understanding of the complex behavior of P-glycoprotein substrates and inhibitors. The results confirm the possibility for multispecific drug interactions of the protein, and are important for elucidating the P-glycoprotein function and ligand interactions. [source] Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous estersFEBS JOURNAL, Issue 10 2008Patrick Masson The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o -nitrophenylacetanilide, o -nitrotrifluorophenylacetanilide and m -(acetamido) N,N,N -trimethylanilinium] and homologous esters (o -nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m -(acetamido) N,N,N -trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (, > , > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric ,cross-talk' between the peripheral anionic site and the catalytic centre. [source] Gas6 and protein SFEBS JOURNAL, Issue 23 2006Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily Gas6 and protein S are two homologous secreted proteins that depend on vitamin K for their execution of a range of biological functions. A discrete subset of these functions is mediated through their binding to and activation of the receptor tyrosine kinases Axl, Sky and Mer. Furthermore, a hallmark of the Gas6,Axl system is the unique ability of Gas6 and protein S to tether their non receptor-binding regions to the negatively charged membranes of apoptotic cells. Numerous studies have shown the Gas6,Axl system to regulate cell survival, proliferation, migration, adhesion and phagocytosis. Consequently, altered activity/expression of its components has been detected in a variety of pathologies such as cancer and vascular, autoimmune and kidney disorders. Moreover, Axl overactivation can equally occur without ligand binding, which has implications for tumorigenesis. Further knowledge of this exquisite ligand,receptor system and the circumstances of its activation should provide the basis for development of novel therapies for the above diseases. [source] A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonistFEBS JOURNAL, Issue 9 2006Brian E. Kane Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity. [source] Tet repressor mutants with altered effector binding and allosteryFEBS JOURNAL, Issue 17 2005Eva-Maria Henßler To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head,core interface on the effector specific TetRi2 variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetRi2 it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures. [source] Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracleFEBS JOURNAL, Issue 13 2002Markus Lezzi The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. [source] Two major Smad pathways in TGF-, superfamily signallingGENES TO CELLS, Issue 12 2002Keiji Miyazawa Members of the transforming growth factor-, (TGF-,) superfamily bind to two different serine/threonine kinase receptors, i.e. type I and type II receptors. Upon ligand binding, type I receptors specifically activate intracellular Smad proteins. R-Smads are direct substrates of type I receptors; Smads 2 and 3 are specifically activated by activin/nodal and TGF-, type I receptors, whereas Smads 1, 5 and 8 are activated by BMP type I receptors. Nearly 30 proteins have been identified as members of the TGF-, superfamily in mammals, and can be classified based on whether they activate activin/TGF-,-specific R-Smads (AR-Smads) or BMP-specific R-Smads (BR-Smads). R-Smads form complexes with Co-Smads and translocate into the nucleus, where they regulate the transcription of target genes. AR-Smads bind to various proteins, including transcription factors and transcriptional co-activators or co-repressors, whereas BR-Smads interact with other proteins less efficiently than AR-Smads. Id proteins are induced by BR-Smads, and play important roles in exhibiting some biological effects of BMPs. Understanding the mechanisms of TGF-, superfamily signalling is thus important for the development of new ways to treat various clinical diseases in which TGF-, superfamily signalling is involved. [source] Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary adenocarcinomas in Syrian golden hamsters contain beta 2-adrenergic receptor single-nucleotide polymorphismsGENES, CHROMOSOMES AND CANCER, Issue 2 2005Thomas Masi Cigarette smoking contributes to the development of lung cancer throughout the world, with cases of pulmonary adenocarcinoma (PAC) the most numerous. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is formed from nicotine, has been demonstrated to cause mutations in genes that affect cell regulation and proliferation. Moreover, NNK has been shown to interact directly with and stimulate beta adrenergic receptor (ADRB) signal transduction pathways. Our goal was to determine whether single-nucleotide polymorphisms (SNPs) in the Adrb2 from PAC tumors were induced in golden hamsters by the injection of NNK. Here we report the cloning and sequencing of Adrb2 clones from either dissected lung tumors from NNK-injected animals or whole-lung tissue from water-injected controls. Both sets of animals contained SNPs; however, we found significantly more SNPs in the Adrb2 from NNK-injected animals than in the controls. The majority of these SNPs were novel, nonsynonymous mutations found in regions of the Adrb2 known to be involved in ligand binding, G-protein coupling, and desensitization/down-regulation. Our data verified the mutagenic effects of NNK as well as demonstrated that this animal model provides an outstanding way of identifying mutations not only in the Adrb2, but also in other genes that may play essential roles in the regulation and growth of pulmonary adenocarcinomas. © 2005 Wiley-Liss, Inc. [source] The major antennal chemosensory protein of red imported fire ant workersINSECT MOLECULAR BIOLOGY, Issue 3 2009D. González Abstract Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si-CSP1 expression, and also showed expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons. [source] Apis mellifera ultraspiracle: cDNA sequence and rapid up-regulation by juvenile hormoneINSECT MOLECULAR BIOLOGY, Issue 5 2004A. R. Barchuk Abstract Two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development including the differentiation of the alternative caste phenotypes of social insects. In addition, JH plays a different role in adult honey bees, acting as a ,behavioural pacemaker'. The functional receptor for 20E is a heterodimer consisting of the ecdysone receptor and ultraspiracle (USP) whereas the identity of the JH receptor remains unknown. We have cloned and sequenced a cDNA encoding Apis mellifera ultraspiracle (AMUSP) and examined its responses to JH. A rapid, but transient up-regulation of the AMUSP messenger is observed in the fat bodies of both queens and workers. AMusp appears to be a single copy gene that produces two transcripts (,4 and ,5 kb) that are differentially expressed in the animal's body. The predicted AMUSP protein shows greater sequence similarity to its orthologues from the vertebrate,crab,tick,locust group than to the dipteran,lepidopteran group. These characteristics and the rapid up-regulation by JH suggest that some of the USP functions in the honey bee may depend on ligand binding. [source] The CAG repeat polymorphism within the androgen receptor gene and maleness,INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2003Michael Zitzmann Summary The androgen testosterone and its metabolite dihydrotestosterone exert their effects on gene expression and thus effect maleness via the androgen receptor (AR). A diverse range of clinical conditions starting with complete androgen insensitivity has been correlated with mutations in the AR. Subtle modulations of the transcriptional activity induced by the AR have also been observed and frequently assigned to a polyglutamine stretch of variable length within the N-terminal domain of the receptor. This stretch is encoded by a variable number of CAG triplets in exon 1 of the AR gene located on the X chromosome. First observations of pathologically elongated AR CAG repeats in patients with X-linked spino-bulbar muscular atrophy showing marked hypoandrogenic traits were supplemented by partially conflicting findings of statistical significance also within the normal range of CAG repeat length: an involvement of prostate tissue, spermatogenesis, bone density, hair growth, cardiovascular risk factors and psychological factors has been demonstrated. The highly polymorphic nature of glutamine residues within the AR protein implies a subtle gradation of androgenicity among individuals within an environment of normal testosterone levels providing relevant ligand binding to ARs. This modulation of androgen effects may be small but continuously present during a man's lifetime and, hence, exerts effects that are measurable in many tissues as various degrees of androgenicity and represents a relevant effector of maleness. It remains to be elucidated whether these insights are important enough to become part of individually useful laboratory assessments. [source] Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin-3 and supports cell migrationINTERNATIONAL JOURNAL OF CANCER, Issue 5 2005Claudia Paret Abstract C4.4A is a member of the Ly6 family, with low homology to uPAR. It has been detected mainly on metastasizing carcinoma cells and proposed to be involved in wound healing. So far, C4.4A has been observed as an orphan receptor, and its functional activity has not been explored. Using recombinant rat C4.4A (rrC4.4A) made in a eukaryotic expression system, we demonstrate by immunohistology that C4.4A ligands are strongly expressed in tissues adjacent to squamous epithelia of, e.g., tongue and esophagus, the expression pattern partly overlapping with laminin (LN) and complementing the C4.4A expression that is found predominantly on the basal layers of squamous epithelium. ELISA screening of several components of the extracellular matrix revealed selective binding of rrC4.4A to LN1 and LN5 and that transfection of the BSp73AS tumor line with C4.4A cDNA (BSp73AS-1B1) promoted LN1 and LN5 binding. Binding of BSp73AS-1B1 to LN5 and, less markedly, LN1 induced spreading, lamellipodia formation and migration. C4.4A also associates with galectin-3 in nontransformed tissues and tumor lines. There is evidence that the association of C4.4A with galectin-3 influences LN adhesion. C4.4A was described originally as a metastasis-associated molecule. Our findings that LN1 and LN5 are C4.4A ligands, that galectin-3 associates with C4.4A and that C4.4A ligand binding confers a migratory phenotype are well in line with the supposed metastasis association. © 2005 Wiley-Liss, Inc. [source] Regulation of signal transduction by glycosylationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Robert S. Haltiwanger The incredible diversity found in cell-surface glycoconjugate structures led researchers over 30 years ago to propose that complexity in carbohydrates must play a role in cellular communication. Recent studies from a number of laboratories have confirmed this hypothesis, demonstrating that cell-surface glycoconjugates play significant roles in signal transduction events. One striking example is the effect of O -fucose modifications on the Notch-signalling pathway. Notch is a cell-surface receptor that is essential for proper development. The extracellular domain of Notch contains up to 36-tandem epidermal growth factor-like (EGF) repeats, many of which are predicted to be modified at putative consensus sequences with O -fucose and O -glucose saccharides. Genetic alterations (by knockout or RNAi methodologies) in the enzyme responsible for the addition of O -fucose to Notch, protein O -fucosyltransferase-1, result in severe, embryonic lethal phenotypes resembling Notch mutants. Thus, O -fucosylation appears to be essential for proper Notch function. Elongation of the O -fucose monosaccharide by the ,1,3- N -acetylglucosaminyltransferase, Fringe, modulates Notch function, either increasing or decreasing response from ligands depending on context. Although it is now clear that O -fucose modifications affect Notch signalling, the molecular mechanism by which this occurs is not known. As an initial step in understanding how O -fucose glycans affect Notch function, we are mapping O -fucose modifications to specific sites on Notch. Already, we have demonstrated that O -fucose modifies one of the EGF repeats involved in ligand binding, suggesting that the sugars may play a role in Notch,ligand interactions. Experiments to test the role of O -fucose modifications at specific sites are in progress. We have also found that Fringe modifies O -fucose on some EGF repeats but not others. Our initial analyses suggest that the basis of this specificity is encoded within the sequences of the individual EGF repeats. Site mapping has also confirmed the presence of O -glucose saccharides on Notch. The evolutionarily conserved, predicted O -glucose sites on Notch are as numerous as those for O -fucose, suggesting that the O -glucose modifications will play an equally important role in Notch biology. We have recently identified an enzymatic activity capable of catalyzing the addition of O -glucose to EGF repeats. Purification of the protein O -glucosyltransferase is underway. These and other results will be discussed. [source] Cooperativity and allostery in haemoglobin functionIUBMB LIFE, Issue 2 2008Chiara Ciaccio Abstract Tetrameric haemoglobins display a cooperative ligand binding behaviour, which has been attributed to the functional interrelationship between multiple ligand binding sites. The quantitative description of this feature was initially carried out with a phenomenological approach, which was limited to the functional effect of the occupancy by a ligand molecule of a binding site on further binding steps. However, subsequent development of structural,functional models for the description of the cooperativity in haemoglobin brought about a much deeper information on the interrelationships between ligand binding at the heme and structural variations occurring in the surrounding free subunits. This approach opened the way to the evolution of the concept of allostery, which is intended as the structural,functional effect exerted by the presence of a ligand in a binding site on other binding sites present in the same molecule. This concept can be applied to either sites for the same ligand (homotropic allostery) and for sites of different ligands (heterotropic allostery). Several models trying to take into account the continuous building up of structural and functional information on the physicochemical properties of haemoglobin have been developed along this line. © 2008 IUBMB IUBMB Life, 60(2): 112,123, 2008 [source] Heme-hemopexin: A ,Chronosteric' heme-proteinIUBMB LIFE, Issue 11 2007Paolo Ascenzi Abstract Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed ,-propeller domains, resembling two thick disks that lock together at a 90° angle. The heme is bound between the two ,-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, ,NO, and cyanide by detaching His213; however, O2 induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates ,NO/O2 and ,NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a ,chronosteric' heme-protein. IUBMB Life, 59: 700-708, 2007 [source] Searching for Neuroglobin's role in the brainIUBMB LIFE, Issue 8-9 2007Karin Nienhaus Abstract Neuroglobin is a small globin that plays an important role in the protection of brain neurons from ischemic and hypoxic injuries. The molecular mechanisms by which Ngb performs its physiological function are still under debate. Suggestions include oxygen storage and delivery, scavenging of NO and/or reactive oxygen species, oxygen sensing and signal transduction. In recent years, the molecular structures of Ngb with carbon monoxide bound to the heme iron and without an exogenous ligand have been solved, and interesting structural changes have been noticed upon ligand binding. Moreover, equilibrium and kinetic properties of the reactions with ligands have been examined in great detail. Here we summarize the molecular properties of Ngb and discuss them in relation to the potential physiological functions. [source] | |||||||||||||||||||||||||||||||||||||