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Extracellular Loop (extracellular + loop)
Kinds of Extracellular Loop Selected AbstractsA tethered ascorbate-norepinephrine compound, 4-UT, displays long-acting adrenergic activity on rabbit aortic smooth muscleDRUG DEVELOPMENT RESEARCH, Issue 5 2008Robert Root-Bernstein Abstract We previously demonstrated that adrenergic and histaminergic receptors have an ascorbic acid (vitamin C) binding site on the first extracellular loop, immediately adjacent to the aminergic binding site. Binding of ascorbate to this site strongly potentiates any sub-maximal dose of an adrenergic or histaminergic compound, significantly increasing its duration of activity. We report here the successful synthesis of a tethered compound that mimics the combined effects of a mixture of ascorbate with norepinephrine. The tethered compound uses a four-unit polyethylene linker to tether ascorbate to norepinephrine. The tethered compound is about tenfold less effective than norepinephrine in stimulating rabbit aortic smooth muscle, but has a very significantly enhanced duration of activity compared with norepinephrine alone and comparable to a mixture of norepinephrine and ascorbate. Additional ascorbate does not enhance the tethered compound's effects and we demonstrate that the compound binds to a synthetic peptide spanning the ascorbate binding site of the receptor. These experiments strongly suggest that the compound binds to both the adrenergic binding site and the ascorbate binding site simultaneously. Tethered compounds with linkers of other lengths did not have these properties. We believe that the synthesis of enhanced adrenergic and histaminergic drugs by tethering them to potentiators such as ascorbate will permit a new class of potential drugs to be created with high specificity and long duration of activity. Drug Dev Res 69:242,250, 2008. © 2008 Wiley-Liss, Inc. [source] Molecular and functional characterization of novel CRFR1 isoforms from the skinFEBS JOURNAL, Issue 13 2004Alexander Pisarchik In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1,, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1, was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1,-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis -elements (CRE, CaRE, SRE, AP1 or NF-,B) demonstrated that only CRFR1, was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4,-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction. [source] A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interactionFEBS JOURNAL, Issue 16 2002Sumandeep K. Sandhu Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl, dependent serotonin uptake, with estimated Km and Vmax values of 436 ± 19 nm and 3.8 ± 0.6 × 10,18 mol·cell·min,1, respectively. The Manduca high affinity Na+/Cl, dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 µm) and cocaine (IC50 = 12.89 µm). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148,152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 µm). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1,146/MasSERT106,587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 µm) compared to the human (IC50 = 0.431 µm) and Manduca serotonin transporters. The chimera MasSERT1,67/hSERT109,630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 µm) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine. [source] Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81,HEPATOLOGY, Issue 6 2007Mirjam B. Zeisel Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture,derived HCV (HCVcc). Anti,SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI,specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81,HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti,SR-BI antibodies and SR-BI,specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV,CD81 interaction. (HEPATOLOGY 2007.) [source] The protein family of glucose transport facilitators: It's not only about glucose after allIUBMB LIFE, Issue 5 2010Robert Augustin Abstract The protein family of facilitative glucose transporters comprises 14 isoforms that share common structural features such as 12 transmembrane domains, N- and C-termini facing the cytoplasm of the cell, and a N-glycosylation side either within the first or fifth extracellular loop. Based on their sequence homology, three classes can be distinguished: class I includes GLUT1-4 and GLUT14, class II the "odd transporters" GLUT5, 7, 9, 11, and class III the "even transporters" GLUT6, 8, 10, 12 and the proton driven myoinositol transporter HMIT (or GLUT13). With the cloning and characterization of the more recent class II and III isoforms, it became apparent that despite their structural similarities, the different isoforms not only show a distinct tissue-specific expression pattern but also show distinct characteristics such as alternative splicing, specific (sub)cellular localization, and affinities for a spectrum of substrates. This review summarizes the current understanding of the physiological role for the various transport facilitators based on human genetically inherited disorders or single-nucleotide polymorphisms and knockout mice models. The emphasis of the review will be on the potential functional role of the more recent isoforms. © 2010 IUBMB IUBMB Life, 62(5): 315,333, 2010 [source] Coupling of Canine Serotonin 5-HT1B and 5-HT1D Receptor Subtypes to the Formation of Inositol Phosphates by Dual Interactions with Endogenous Gi/o and Recombinant G,15 ProteinsJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Thierry Wurch Abstract: Molecular cloning and expression of canine (ca) serotonin 5-HT1B and ca 5-HT1D receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT1D receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln189 residue in the second extracellular loop of the ca 5-HT1D receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT1B and ca 5-HT1D receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC50 = 4.9 nM) the inositol phosphate formation at ca 5-HT1D receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 ,M zolmitriptan) could be detected for the ca 5-HT1B receptor at a similar expression level. In contrast, both ca 5-HT1B and ca 5-HT1D receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 ,M zolmitriptan) upon co-expression with a mouse (m) G,15 protein. PTX treatment and co-expression with a ,-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G,15 protein-dependent ca 5-HT1B and ca 5-HT1D receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate ,, subunits of endogenous Gi/o proteins besides their coupling to recombinant m G,15 protein. [source] Towards an understanding of organic anion transporters: Structure,function relationshipsMEDICINAL RESEARCH REVIEWS, Issue 6 2004Guofeng You Abstract Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including anti-viral drugs, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. The activities of OATs are directly linked to drug toxicity and drug,drug interactions. So far, four members of the OAT family have been identified: OAT1, OAT2, OAT3, and OAT4. These transporters share several common structural features including 12 transmembrane domains, multiple glycosylation sites localized in the first extracellular loop between transmembrane domains 1 and 2, and multiple phosphorylation sites present in the intracellular loop between transmembrane domains 6 and 7, and in the carboxyl terminus. The impact of these structural features on the function of these transporters has just begun to be explored. In the present review, the author will summarize recent progress made from her laboratory as well as from others, on the molecular characterization of the structure,function relationships of OATs, including particular amino acid residues/regions of the transporter protein ("molecular domains") that potentially determine transport characteristics. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 6, 762,774, 2004 [source] Cloning, sequence, and function analyses of giant panda (Ailuropoda melanoleuca) CD9 gene,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2008Yang Tang Abstract CD9 is a member of the tetraspanin family proteins and has recently been shown to be essential for sperm,oocyte fusion in mice. The giant panda (Ailuropoda melanoleuca) CD9 (gpCD9) cDNA was amplified for the first time by RT-PCR from ovary total RNA and cloned, sequenced and analyzed. The result revealed that the open reading frame (ORF) of gpCD9 was 681 bp, which has the same length as that of mouse. Sequence analysis and structure prediction displayed that the amino acid sequence of gpCD9 is over 80% identity to those of mammals with the conserved structures, including the four transmembrane domains (TM) and certain characteristic residues. The results of sperm,egg fusion experiments demonstrated that giant panda CD9 large extracellular loop (LEL) significantly inhibited (P,<,0.05) the mouse gamete fusion when the recombinant protein was added. However, when three amino acid residues TVT (173,175) of the gpCD9 were mutated to AAA, the large extracellular loop (LELM) of mutated protein was rarely inhibiting the gamete fusion of mice. Our results may be useful in improving an insight into understanding the potential mechanism of gamete fusion and genetic characteristics of giant panda. Mol. Reprod. Dev. 75: 1418,1425, 2008. © 2008 Wiley-Liss, Inc. [source] Three monophyletic superfamilies account for the majority of the known glycosyltransferasesPROTEIN SCIENCE, Issue 7 2003Jing Liu Abstract Sixty-five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high-sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern-type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT-B superfamily; (2) a nucleoside-diphosphosugar transferase (GT-A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT-C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout-velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein. [source] Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchangeTHE JOURNAL OF PHYSIOLOGY, Issue 18 2010Kenneth B. Gagnon Na+,K+,2Cl, cotransport (NKCC) mediates the movement of two Cl, ions for one Na+ and one K+ ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl, uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K+ uptake with only a minimal increase in bumetanide-sensitive Cl, uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K+/K+ and Cl,/Cl, exchange. Although significant stimulation of K+/K+ exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K+/K+ exchange. [source] Substrate interactions of the electroneutral Na+ -coupled inorganic phosphate cotransporter (NaPi-IIc)THE JOURNAL OF PHYSIOLOGY, Issue 17 2009Chiara Ghezzi The SLC34 solute carrier family comprises the electrogenic NaPi-IIa/b and the electroneutral NaPi-IIc, which display Na+ : Pi cotransport stoichiometries of 3 : 1 and 2 : 1, respectively. We previously proposed that NaPi-IIc lacks one of the three Na+ interaction sites hypothesised for the electrogenic isoforms, but, unlike NaPi-IIa/b, its substrate binding order is undetermined. By expressing NaPi-IIc in Xenopus oocytes, isotope influx and efflux assays gave results consistent with Na+ being the first and last substrate to bind. To further investigate substrate interactions, we applied a fluorometry-based technique that uses site-specific labelling with a fluorophore to characterize substrate-induced conformational changes. A novel Cys was introduced in the third extracellular loop of NaPi-IIc that could be labelled with a reporter fluorophore (MTS-TAMRA). Although labelling resulted in suppression of cotransport as previously reported for the electrogenic isoforms, changes in fluorescence were induced by changes in extracellular Na+ concentration in the absence of Pi and by changes in extracellular Pi concentration in presence of Na+. These data, combined with 32P uptake data, also support a binding scheme in which Na+ is the first substrate to interact. Moreover, the apparent Pi affinity from fluorometry agreed with that from 32P uptake, confirming the applicability of the fluorometric technique for kinetic studies of electroneutral carriers. Analysis of the fluorescence data showed that like the electrogenic NaPi-IIb, 2 Na+ ions interact cooperatively with NaPi-IIc before Pi binding, which implies that only one of these is translocated. This result provides compelling evidence that SLC34 proteins share common motifs for substrate interaction and that cotransport and substrate binding stoichiometries are not necessarily equivalent. [source] Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannelTHE JOURNAL OF PHYSIOLOGY, Issue 10 2008Seunghoon Oh Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32*43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter. [source] Antimuscarinic antibodies in primary Sjögren's syndrome reversibly inhibit the mechanism of fluid secretion by human submandibular salivary acinar cellsARTHRITIS & RHEUMATISM, Issue 4 2006L. J. Dawson Objective Sjögren's syndrome (SS) is an autoimmune condition affecting salivary glands, for which a clearly defined pathogenic autoantibody has yet to be identified. Autoantibodies that bind to the muscarinic M3 receptors (M3R), which regulate fluid secretion in salivary glands, have been proposed in this context. However, there are no previous data that directly show antisecretory activity. This study was undertaken to investigate and characterize the antisecretory activity of anti-M3R. Methods Microfluorimetric Ca2+ imaging and patch clamp electrophysiologic techniques were used to measure the secretagogue-evoked increase in [Ca2+]i and consequent activation of Ca2+ -dependent ion channels in individual mouse and human submandibular acinar cells. Together, these techniques form a sensitive bioassay that was used to determine whether IgG isolated from patients with primary SS and from control subjects has antisecretory activity. Results IgG (2 mg/ml) from patients with primary SS reduced the carbachol-evoked increase in [Ca2+]i in both mouse and human acinar cells by ,50%. IgG from control subjects had no effect on the Ca2+ signal. Furthermore, the inhibitory action of primary SS patient IgG on the Ca2+ signal was acutely reversible. We repeated our observations using rabbit serum containing antibodies raised against the second extracellular loop of M3R and found an identical pattern of acutely reversible inhibition. Anti-M3R,positive serum had no effect on Ca2+ -dependent ion channel activation evoked by the direct intracellular infusion of inositol 1,4,5-triphosphate. Conclusion These observations show for the first time that IgG from patients with primary SS contains autoantibodies capable of damaging saliva production and contributing to xerostomia. The unusual but not unprecedented acute reversibility of the effects of anti-M3 autoantibodies is the subject of further research. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Karl Brillet As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5,Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3,Å resolution using one crystal of ShuA-Pb, and at 3.2,Å resolution at an energy remote from the Pb,M absorption edges for phasing on PROXIMA-1 at SOLEIL. [source] Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT1A receptorBIOPOLYMERS, Issue 1 2002Roberto K. Salinas Abstract Homology modeling of the angiotensin II AT1A receptor based on rhodopsin,s crystal structure has assigned the 92,100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane-bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and 1H-NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp3,Pro4 bond. A positive band at 226 nm in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different ,-turns. Chemical shift changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing ,-turns, predicted for this sequence in the whole receptor. Increases in Trp3 fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well as lipid phase packing. Studies with water-soluble and membrane-bound fluorescence quenchers demonstrated that Trp3 is located close to the water,membrane interface. The results are discussed with regard to possible implications in receptor folding and function. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 21,31, 2002 [source] Therapeutic antitumor efficacy of monoclonal antibody against Claudin-4 for pancreatic and ovarian cancersCANCER SCIENCE, Issue 9 2009Masayo Suzuki Claudin-4 (CLDN4) is a tetraspanin transmembrane protein of tight junction structure and is highly expressed in pancreatic and ovarian cancers. In this study, we aimed to generate an anti-Claudin-4 monoclonal antibody (mAb) and evaluate its antitumor efficacy in vitro and in vivo. To isolate specific mAb, we generated CLDN3, 4, 5, 6, and 9, expressing Chinese hamster ovary (CHO) cells, and then used them as positive and negative targets through cell-based screening. As a result, we succeeded in isolating KM3900 (IgG2a), which specifically bound to CLDN4, from BXSB mice immunized with pancreatic cancer cells. Immunoprecipitation and flow cytometry analysis revealed that KM3900 recognized the conformational structure and bound to extracellular loop 2 of CLDN4. Furthermore, binding of KM3900 was detected on CLDN4-expressing pancreatic and ovarian cancer cells, but not on negative cells. Next, we made the mouse,human chimeric IgG1 (KM3934) and evaluated its antitumor efficacy. KM3934 induced dose-dependent antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and significantly inhibited tumor growth in MCAS or CFPAC-1 xenograft SCID mice in vivo (P < 0.05). These results suggest that mAb therapy against CLDN4 is promising for pancreatic and ovarian cancers. (Cancer Sci 2009; 100: 1623,1630) [source] Synthesis and secondary structure of loop 4 of myelin proteolipid protein: effect of a point mutation found in Pelizaeus-Merzbacher diseaseCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2005E. Trifilieff Abstract:, To study the effects of a point mutation found in Pelizaeus-Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181,230)Pro215 and one mutant PLP(181,230)Ser215 with regioselective formation of the two disulphide bridges Cys200 -Cys219 and Cys183 -Cys227. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro215 the first bridge Cys200,Cys219 was obtained after air oxidation, but in peptide Ser215 because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys183,Cys227 was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)-protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of , -helix and , -sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence. [source] The Predicted 3D Structures of the Human M1 Muscarinic Acetylcholine Receptor with Agonist or Antagonist BoundCHEMMEDCHEM, Issue 8 2006Joyce Yao-chun Peng Abstract The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand,receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices,3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp,157(4) contributes directly to antagonist binding, whereas Pro,159(4) provides an indirect conformational switch to position Trp,157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp,157(4) and Pro,159(4) not previously explained by homology models. We also found that Asp,105(3) and aromatic residues Tyr,381(6), Tyr,404(7), and Tyr,408(7) are critical for binding the quaternary ammonium head group of the ligand through cation,, interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp,105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr,106(3), Trp,157(4), Phe,197(5), Trp,378(6), and Tyr,381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp,164(4), Tyr,179(extracellular loop,2), and Phe,390(6) located at the extracellular end of the TMs. We find that Asn,382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser,109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation. [source] CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2003Georgina Xanthou Abstract The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-,-inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We alsodemonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3,CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo. [source] Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2,CD81,Claudin-1 associations,HEPATOLOGY, Issue 4 2010Sophie E. Krieger The tight junction protein claudin-1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry,the first step of viral infection. Due to the lack of neutralizing anti-CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface,expressed CLDN1 specifically inhibit HCV infection in a dose-dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti-CLDN1 and anti-CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti-CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1-E2 interaction. Using fluorescent-labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti-CLDN1 antibodies inhibit CD81-CLDN1 association. In contrast, CLDN1-CLDN1 and CD81-CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81-CLDN1 interactions. Conclusion: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2-CD81-CLDN1 interactions. (HEPATOLOGY 2010.) [source] PTCH mutations: distribution and analyses,HUMAN MUTATION, Issue 3 2006Erika Lindström Abstract Mutations in the PTCH (PTCH1) gene are the underlying cause of nevoid basal cell carcinoma syndrome (NBCCS), and are also found in many different sporadic tumors in which PTCH is thought to act as a tumor suppressor gene. To investigate the distribution pattern of these mutations in tumors and NBCCS, we analyzed 284 mutations and 48 SNPs located in the PTCH gene that were compiled from our PTCH mutation database. We found that the PTCH mutations were mainly clustered into the predicted two large extracellular loops and the large intracellular loop. The SNPs appeared to be clustered around the sterol sensing domain and the second half of the protein. The NBCCS cases and each class of tumor analyzed revealed a different distribution of the mutations in the various PTCH domains. Moreover, the types of mutations were also unique for the different groups. Finally, the PTCH gene harbors mutational hot spot residues and regions, including a slippage-sensitive sequence in the N-terminus. Hum Mutat 27(3), 215,219, 2006. © 2006 Wiley-Liss, Inc. [source] A description of the structural determination procedures of a gap junction channel at 3.5,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Michihiro Suga Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5,Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. [source] Dissecting membrane protein architecture: An annotation of structural complexityBIOPOLYMERS, Issue 10 2009Jaime Arce Abstract ,-Helical membrane proteins exist in an anisotropic environment which strongly influences their folding, stability, and architecture, which is far more complex than a simple bundle of transmembrane helices, notably due to helix deformations, prosthetic groups and extramembrane structures. However, the role and the distribution of such heterogeneity in the supra molecular organization of membrane proteins remains poorly investigated. Using a nonredundant subset of ,-helical membrane proteins, we have annotated and analyze the statistics of several types of new elements such as incomplete helices, intramembrane loops, helical extensions of helical transmembrane domains, extracellular loops, and helices lying parallel to the membrane surface. The relevance of the annotation scheme was studied using residue composition, statistics, physical chemistry, and symmetry of their distribution in relation to the immediate membrane environment. Calculation of hydrophobicity using different scales show that different structural elements appear to have affinities coherent with their position in the membrane. Examination of the annotation scheme suggests that there is considerable information content in the amino acid compositions of the different elements suggesting that it might be useful for structural prediction. More importantly, the proposed annotation will help to decipher the complex hierarchy of interactions involved in membrane protein architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 815,829, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | ||||||||||||||||||||||||||||