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Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.
Mousa JJ, Binshtein E, Human S, Fong RH, Alvarado G, Doranz BJ, Moore ML, Ohi MD, Crowe JE
(2018) PLoS Pathog 14: e1006837
MeSH Terms: Amino Acid Sequence, Amino Acid Substitution, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Antibody Specificity, Binding Sites, Antibody, Binding, Competitive, Cross Reactions, Epitope Mapping, Epitopes, Humans, Kinetics, Metapneumovirus, Microscopy, Electron, Mutation, Recombinant Proteins, Respiratory Syncytial Virus, Human, Viral Fusion Proteins
Show Abstract · Added March 3, 2020
Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.
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Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
Zurawski Z, Page B, Chicka MC, Brindley RL, Wells CA, Preininger AM, Hyde K, Gilbert JA, Cruz-Rodriguez O, Currie KPM, Chapman ER, Alford S, Hamm HE
(2017) J Biol Chem 292: 12165-12177
MeSH Terms: Animals, Binding, Competitive, Calcium Signaling, Cattle, Cell Line, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Humans, Lipid Bilayers, Liposomes, Membrane Fusion, Models, Molecular, Mutation, Nerve Tissue Proteins, Peptide Fragments, Protein Conformation, Protein Interaction Domains and Motifs, Protein Multimerization, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Synaptosomal-Associated Protein 25, Synaptotagmin I, Syntaxin 1
Show Abstract · Added July 12, 2017
G-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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24 MeSH Terms
The pattern recognition reagents RAGE VC1 and peptide p5 share common binding sites and exhibit specific reactivity with AA amyloid in mice.
Kennel SJ, Williams A, Stuckey A, Richey T, Wooliver C, Chazin W, Stern DA, Martin EB, Wall JS
(2016) Amyloid 23: 8-16
MeSH Terms: Amyloidosis, Animals, Binding, Competitive, Iodine Radioisotopes, Mice, Inbred BALB C, Mice, Transgenic, Peptides, Protein Binding, Radiography, Dual-Energy Scanned Projection, Radiopharmaceuticals, Receptor for Advanced Glycation End Products, Recombinant Proteins, Serum Amyloid A Protein, Technetium, Tissue Distribution, Tomography, X-Ray Computed
Show Abstract · Added March 11, 2020
UNLABELLED - In the US, there remains a need to develop a clinical method for imaging amyloid load in patients with systemic, visceral amyloidosis. The receptor for advanced glycation end products (RAGE), which exists as a transmembrane receptor and soluble variant, is found associated with a number of amyloid deposits in man. It is unclear whether amyloid-associated RAGE is the membrane or soluble form; however, given the affinity of RAGE for amyloid, we have examined the ability of soluble RAGE VC1 to specifically localize with systemic AA amyloid in mice. We further compared the reactivity of RAGE VC1 with that of the synthetic, amyloid-reactive peptide p5.
METHODS - Binding of radiolabeled RAGE VC1 and p5 to synthetic amyloid fibrils was evaluated using in vitro "pulldown" assays in the presence or absence of RAGE ligands. Radioiodinated RAGE VC1 and technetium-99 m-labeled p5 were studied in mice with systemic AA amyloidosis using dual-energy SPECT/CT imaging, biodistribution and microautoradiography.
RESULTS - Soluble RAGE VC1 competed with radioiodinated peptide p5 for binding to rVλ6Wil, Aβ (1-40) and IAPP fibrils but not with the higher affinity peptide, p5R. Pre-incubation with AGE-BSA abrogated binding of VC1 and p5 to rVλ6Wil fibrils. Dual-energy SPECT/CT images and quantitative tissue biodistribution data showed that soluble RAGE VC1 specifically bound AA amyloid-laden organs in mice as effectively as peptide p5. Furthermore, microautoradiography confirmed that RAGE VC1 bound specifically to areas of Congo red-positive amyloid in mouse tissues but not in comparable tissues from control WT mice.
CONCLUSION - Soluble RAGE VC1 and peptide p5 have similar ligand binding properties and specifically localize with visceral AA amyloid deposits in mice.
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Competition and allostery govern substrate selectivity of cyclooxygenase-2.
Mitchener MM, Hermanson DJ, Shockley EM, Brown HA, Lindsley CW, Reese J, Rouzer CA, Lopez CF, Marnett LJ
(2015) Proc Natl Acad Sci U S A 112: 12366-71
MeSH Terms: Algorithms, Allosteric Regulation, Allosteric Site, Animals, Arachidonic Acid, Arachidonic Acids, Binding, Competitive, Catalytic Domain, Cell Line, Computer Simulation, Cyclooxygenase 2, Endocannabinoids, Glycerides, Kinetics, Macrophages, Mice, Oxidation-Reduction, Prostaglandins, Protein Binding, Protein Multimerization, Sf9 Cells, Spodoptera, Substrate Specificity, Zymosan
Show Abstract · Added February 15, 2016
Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.
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Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.
Kim D, Cha GS, Nagy LD, Yun CH, Guengerich FP
(2014) Biochemistry 53: 6161-72
MeSH Terms: Algorithms, Binding, Competitive, Biocatalysis, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System, Cytochromes b5, Deuterium, Electron Transport, Ferric Compounds, Ferrous Compounds, Humans, Hydroxylation, Kinetics, Lauric Acids, Models, Chemical, Models, Molecular, Oxidation-Reduction, Protein Binding, Protein Structure, Tertiary, Substrate Specificity, Tritium
Show Abstract · Added January 20, 2015
Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.
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21 MeSH Terms
Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254).
Turlington M, Noetzel MJ, Chun A, Zhou Y, Gogliotti RD, Nguyen ED, Gregory KJ, Vinson PN, Rook JM, Gogi KK, Xiang Z, Bridges TM, Daniels JS, Jones C, Niswender CM, Meiler J, Conn PJ, Lindsley CW, Stauffer SR
(2013) J Med Chem 56: 7976-96
MeSH Terms: Acetylene, Allosteric Regulation, Amides, Animals, Binding, Competitive, Cell Membrane, Drug Discovery, Excitatory Postsynaptic Potentials, HEK293 Cells, Hippocampus, Humans, Male, Models, Chemical, Models, Molecular, Molecular Structure, Picolinic Acids, Protein Structure, Tertiary, Radioligand Assay, Rats, Rats, Sprague-Dawley, Receptor, Metabotropic Glutamate 5, Structure-Activity Relationship
Show Abstract · Added January 24, 2015
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.
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22 MeSH Terms
JNK3 enzyme binding to arrestin-3 differentially affects the recruitment of upstream mitogen-activated protein (MAP) kinase kinases.
Zhan X, Kaoud TS, Kook S, Dalby KN, Gurevich VV
(2013) J Biol Chem 288: 28535-47
MeSH Terms: Animals, Arrestins, Binding, Competitive, COS Cells, Chlorocebus aethiops, Humans, MAP Kinase Kinase 4, MAP Kinase Kinase 7, Mice, Mitogen-Activated Protein Kinase 10, Phosphorylation, Protein Binding, Substrate Specificity
Show Abstract · Added December 10, 2013
Arrestin-3 was previously shown to bind JNK3α2, MKK4, and ASK1. However, full JNK3α2 activation requires phosphorylation by both MKK4 and MKK7. Using purified proteins we show that arrestin-3 directly interacts with MKK7 and promotes JNK3α2 phosphorylation by both MKK4 and MKK7 in vitro as well as in intact cells. The binding of JNK3α2 promotes an arrestin-3 interaction with MKK4 while reducing its binding to MKK7. Interestingly, the arrestin-3 concentration optimal for scaffolding the MKK7-JNK3α2 module is ∼10-fold higher than for the MKK4-JNK3α2 module. The data provide a mechanistic basis for arrestin-3-dependent activation of JNK3α2. The opposite effects of JNK3α2 on arrestin-3 interactions with MKK4 and MKK7 is the first demonstration that the kinase components in mammalian MAPK cascades regulate each other's interactions with a scaffold protein. The results show how signaling outcomes can be affected by the relative expression of scaffolding proteins and components of signaling cascades that they assemble.
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13 MeSH Terms
Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.
Li F, Xia Y, Meiler J, Ferguson-Miller S
(2013) Biochemistry 52: 5884-99
MeSH Terms: Amino Acid Sequence, Apoptosis, Binding, Competitive, Carrier Proteins, Cholesterol, Cryoelectron Microscopy, Crystallization, Isoquinolines, Membrane Proteins, Mitochondrial Membranes, Protein Multimerization, Protoporphyrins, Receptors, GABA-A, Rhodobacter sphaeroides, Sequence Alignment
Show Abstract · Added January 24, 2015
Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.
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15 MeSH Terms
Quantum dot approaches for target-based drug screening and multiplexed active biosensing.
Kovtun O, Arzeta-Ferrer X, Rosenthal SJ
(2013) Nanoscale 5: 12072-81
MeSH Terms: Animals, Binding, Competitive, Biosensing Techniques, Drug Evaluation, Preclinical, Endpoint Determination, Humans, Molecular Targeted Therapy, Quantum Dots, Surface Properties
Show Abstract · Added May 27, 2014
Biomolecule detection using quantum dots (Qdots), nanometer-sized semiconductor crystals, effectively addresses the limitations associated with conventional optical and biochemical techniques, as Qdots offer several key advantages over traditional fluorophores. In this minireview, we discuss the role of Qdots as a central nanoscaffold for the polyvalent assembly of multifunctional biomolecular probes and describe recent advances in Qdot-based biorecognition. Specifically, we focus on Qdot applications in target-based, drug screening assays and real-time active biosensing of cellular processes.
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Approaches for probing allosteric interactions at 7 transmembrane spanning receptors.
Klein MT, Vinson PN, Niswender CM
(2013) Prog Mol Biol Transl Sci 115: 1-59
MeSH Terms: Allosteric Regulation, Animals, Binding, Competitive, Biological Assay, Humans, Kinetics, Pharmaceutical Preparations, Receptors, Cell Surface
Show Abstract · Added September 12, 2013
In recent years, allosteric modulation of 7 transmembrane spanning receptors (7TMRs) has become a highly productive and exciting field of receptor pharmacology and drug discovery efforts. Positive and negative allosteric modulators (PAMs and NAMs, respectively) present a number of pharmacological and therapeutic advantages over conventional orthosteric ligands, including improved receptor-subtype selectivity, a lower propensity to induce receptor desensitization, the preservation of endogenous temporal and spatial activation of receptors, greater chemical flexibility for optimization of drug metabolism and pharmacokinetic parameters, and saturability of effect at target receptors, thus improving safety concerns and risk of overdose. Additionally, the relatively new concept of allosteric modulator-mediated receptor signal bias opens up a number of intriguing possibilities for PAMs, NAMs, and allosteric agonists, including the potential to selectively activate therapeutically beneficial signaling cascades, which could yield a superior tissue selectivity and side effect profile of allosteric modulators. However, there are a number of considerations and caveats that must be addressed when screening for and characterizing the properties of 7TMR allosteric modulators. Mode of pharmacology, methodology used to monitor receptor activity, detection of appropriate downstream analytes, selection of orthosteric probe, and assay time-course must all be considered when implementing any high-throughput screening campaign or when characterizing the properties of active compounds. Yet compared to conventional agonist/antagonist drug discovery programs, these elements of assay design are often a great deal more complicated when working with 7TMRs allosteric modulators. Moreover, for classical pharmacological methodologies and analyses, like radioligand binding and the assessment of compound affinity, the properties of allosteric modulators yield data that are more nuanced than orthosteric ligand-receptor interactions. In this review, we discuss the current methodologies being used to identify and characterize allosteric modulators, lending insight into the approaches that have been most successful in accurately and robustly identifying hit compounds. New label-free technologies capable of detecting phenotypic cellular changes in response to receptor activation are powerful tools well suited for assessing subtle or potentially masked cellular responses to allosteric modulation of 7TMRs. Allosteric modulator-induced receptor signal bias and the assay systems available to probe the various downstream signaling outcomes of receptor activation are also discussed.
Copyright © 2013 Elsevier Inc. All rights reserved.
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8 MeSH Terms