Home About us Contact
|
Home About us Contact | ||
|
|
||
Ligand Specificity (ligand + specificity)
Selected AbstractsDirected Evolution of Orthogonal Ligand Specificity in a Single Scaffold,ANGEWANDTE CHEMIE, Issue 42 2009Michael J. McLachlan Zwei hochempfindliche Ligand-Rezeptor-Paare, die auf einem einzigen Proteingerüst beruhen, sind zum nativen Ligand-Rezeptor-Paar und zueinander vollständig orthogonal. Alle drei Rezeptoren werden durch ihre jeweiligen Liganden (rote Elemente im Bild) spezifisch aktiviert, wenn mit ihnen die Expression von grün fluoreszierendem (obere Reihe), mCherry- (mittlere Reihe) und gelb fluoreszierendem Protein (untere Reihe) in Hefe nachgewiesen werden soll. [source] Chimeric receptor analyses of the interactions of the ectodomains of ErbB-1 with epidermal growth factor and of those of ErbB-4 with neuregulinFEBS JOURNAL, Issue 9 2002Jae-Hoon Kim A series of chimeric receptors was generated between the epidermal growth factor (EGF) receptor, ErbB-1, and its homologue, ErbB-4, to investigate the roles of the extracellular domains (I,IV) in the ligand specificities. As compared with ErbB-1 and the chimeras with both domains I and III of ErbB-1, the chimeras with only one of these domains exhibited reduced binding of 125I-labeled EGF. Particularly, the contribution of domain III was appreciably larger than that of domain I of ErbB-1 in 125I-labeled EGF binding. Nevertheless, the chimeras with domain III of ErbB-1 and domain I of ErbB-4 were prevented from binding to 125I-labeled EGF competitively by the ErbB-4 ligand, neuregulin (NRG). On the other hand, NRG did not compete with 125I-labeled EGF for binding to the chimeras with the ErbB-1 domain I and the ErbB-4 domain III. Therefore, NRG binding to ErbB-4 depends much more on domain I than on domain III. With respect to autophosphorylation and subsequent ERK activation, EGF activated the chimeras with either domain I or III of ErbB-1. In contrast, NRG activated the chimeras with the ErbB-4 domain I and the ErbB-1 domain III, but not those with the ErbB-1 domain,I and the ErbB-4 domain III. Therefore, the relative contributions between domains I and III of ErbB-4 in the NRG signaling are different from those of ErbB-1 in the EGF signaling. [source] Analyses for binding of the transferrin family of proteins to the transferrin receptor 2BRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2004Hiroshi Kawabata Summary Transferrin receptor 2, (TfR2,), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2, may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2, using human transferrin receptor 1 (TfR1) and TfR2, -stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2, interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2, interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2,, the RGD sequence in the extracellular domain of TfR2, was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2, (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2, share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism. [source] Molecular determinants of ligand specificity in family 11 carbohydrate binding modules , an NMR, X-ray crystallography and computational chemistry approachFEBS JOURNAL, Issue 10 2008Aldino Viegas The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. [source] CYP3A4 and pregnane X receptor humanized miceJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2007Frank J. Gonzalez Abstract Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR -humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16,-carbonitrile. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:158,162, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20173 [source] Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responsesJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Robert J. Romanelli Abstract The trafficking of receptor tyrosine kinases (RTKs) to distinct subcellular locations is essential for the specificity and fidelity of signal transduction and biological responses. This is particularly important in the PNS and CNS in which RTKs mediate key events in the development and maintenance of neurons and glia through a wide range of neural processes, including survival, proliferation, differentiation, neurite outgrowth, and synaptogenesis. The mechanisms that regulate the targeting of RTKs to their subcellular destinations for appropriate signal transduction, however, are still elusive. In this review, we discuss evidence for the spatial organization of signaling machinery into distinct subcellular compartments, as well as the role for ligand specificity, receptor sorting signals, and lipid raft microdomains in RTK targeting and the resultant cellular responses in neural cells. [source] Analysis of ligand binding to a ribose biosensor using site-directed mutagenesis and fluorescence spectroscopyPROTEIN SCIENCE, Issue 3 2007Natalie C. Vercillo Abstract Computational design of proteins with altered ligand specificity is an emerging method for the creation of new biosensing systems. In this work, we investigated the outcome of site-directed mutagenesis on the Escherichia coli ribose binding protein (RBP), which is frequently used as a design scaffold for computational searches. A ribose biosensor was first constructed whereby an environmentally sensitive fluorescent probe was covalently attached to RBP at position S265C. This protein conjugate displayed a 54% decrease in emission intensity upon the addition of saturating ribose concentrations and exhibited an apparent dissociation constant (Kd) of 3.4 ,M. Site-directed mutants within the RBP binding pocket were created and examined for ribose binding ability and overall structural stability. Because as many as 12 mutations are needed to alter ligand specificity in RBP, we measured the effect of single and multiple alanine mutations on stability and signal transduction potential of the ribose biosensor. Single alanine mutations had significant impact on both stability and signaling. Mutations of N190A and F214A each produced melting temperatures >8°C below those observed for the wild-type protein. Residue Q235, located in the hinge region of RBP, appeared to be a hot spot for global protein stability as well. Additional single alanine mutations demonstrated as much as 200-fold increase in apparent Kd but retained overall protein stability. The data collected from this study may be incorporated into design algorithms to help create more stable biosensors and optimize signal transduction properties for a variety of important analytes. [source] Analyses for binding of the transferrin family of proteins to the transferrin receptor 2BRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2004Hiroshi Kawabata Summary Transferrin receptor 2, (TfR2,), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2, may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2, using human transferrin receptor 1 (TfR1) and TfR2, -stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2, interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2, interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2,, the RGD sequence in the extracellular domain of TfR2, was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2, (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2, share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism. [source] Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesisCANCER SCIENCE, Issue 5 2004Reiji Kannagi Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewisa/x determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system. [source] AN S296R MUTATION IN THE HUMAN ANDROGEN RECEPTOR CAUSES ACTIVATION OF THE RECEPTOR BY NON-ANDROGENIC STEROIDS AND STRONGER INHIBITION BY THE NUCLEAR RECEPTOR COREPRESSOR N-coRCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2008Yi-Dong Li SUMMARY 1Mutation of the androgen receptor (AR) is believed to contribute to androgen-independent growth of prostate cancer. In the present study, we examined the functional changes associated with the novel somatic mutation S296R in the N-terminal domain of the AR identified from one recurrent prostate cancer sample. 2The results indicate that the S296R mutation does not differ obviously from the wild-type AR in its ability to bind the synthetic androgen methyltrienolone, or in its transcriptional activity induced by dihydrotestosterone (DHT) in the absence or presence of the overexpression of coactivators (steroid receptor coactivator-1, transcription intermediary factor-2, cAMP response element-binding protein-binding protein and p300). However, S296R was found to differ from wild-type AR in that its transcriptional activity could be activated by high concentrations (1 µmol/L) of 17,-oestradiol and progesterone and its transactivity induced by DHT was more obviously inhibited by overexpression of the nuclear receptor corepressor N-coR in CV-1 cells. 3These findings indicate that a point mutation (S296R) in the N-terminal domain of the AR can decrease the ligand specificity of the AR and alter the interaction between S296R and the corepressor N-coR. [source] | |||||||||||||