Analysis Of Ligand Binding Research Articles

Single Cell Analysis of Ligand Binding and Complex Formation of Interleukin-4 Receptor Subunits

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.

Both AT1and AT2receptors mediate proliferation and migration of porcine vascular smooth muscle cells

Angiotensin receptor antagonists have shown clinical promise in modulating vascular disease, in part by limiting smooth muscle cell proliferation and migration. The majority of studies examining the contribution of these receptors have been undertaken in cells derived from rat aorta, which primarily express the ANG II type 1 (AT(1)) receptor. This investigation studied the relative contribution of AT(1) and ANG II type 2 (AT(2)) receptors to the mitogenic program of porcine smooth muscle cells. Smooth muscle cells were derived from porcine coronary artery explants. The presence of both AT(1) and AT(2) receptors was demonstrated through ligand binding and RT-PCR analysis. Biochemical and cellular markers of proliferation were monitored in the presence of selective receptor antagonists. Smooth muscle cell migration was measured using both wound healing and Boyden chamber migration assays. Visualization of the AT(1) and AT(2) receptors in growing and quiescent porcine smooth muscle cells with epifluorescence microscopy demonstrated that their subcellular distribution varied with growth state. An examination with several growth assays revealed that both AT(1)-specific losartan and AT(2)-specific PD-123319 receptor antagonists inhibited ANG II-stimulated RNA and DNA synthesis, PCNA expression, and hyperplasia. ANG II induced both directional and nondirectional cell migration. AT(1) receptor antagonist treatment significantly decreased ANG II-induced directional migration only, whereas AT(2) receptor antagonist treatment significantly reduced both modes of migration. Interestingly, the focal adhesion kinase inhibitor PF-573228 also blocked migration but not proliferation. Furthermore, focal adhesion kinase activation in response to ANG II was prevented only by PD-123319, indicating that this activation is downstream of the AT(2) receptor. The observed role of the AT(2) receptor in ANG II-induced migration was confirmed with smooth muscle cells depleted of the AT(2) receptor with short hairpin RNA treatment.

A DFT study of determination of the reactive sites of the acetylcholine and its agonists: In the gas phase and dielectric medium

AbstractThe reactive behavior of acetylcholine and its agonist molecules have been investigated using B3LYP hybrid density functional method at the 6‐311++G** basis set level, in the gas phase and aqueous phase. The calculations have been performed to obtain optimized geometries, relative reactivities, net atomic charges, HOMO, and LUMO energies. The solvent effect has been analyzed by using the continuum model (IPCM) and, the obtained results have shown that the all molecules have been stabilized more by solvent dielectric constant. For Ach and its analogues, it has been very well known that esteratic site and quaternary ammonium group which have reflected the difference in biological activity have been the two of the most important active site for interactions between molecule and its receptor. The structures of these analogues have provided an essential foundation for subsequent structure‐activity analysis of ligand binding at acetylcholine receptors, neuronal uptake inhibitors and transporters. Molecular modeling predictions will be important initial steps toward the development of novel pharmaceuticals in the fight acetylcholine‐related neurological disorders. This work is therefore expected to facilitate the design and development of new biologically active Ach analogues to treat Ach‐related neurological disorders and, specially is used to qualitative understanding of the reactivity and related properties and, so on can be used to a preselection of new ligands which at the moment is taken essentially from empirical knowledge. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Abstract LB-397: Functional characterization of IGF-1R antibodies and possible implications for clinical safety and efficacy

Abstract Background This is the first study performing a head to head comparison of monoclonal IGF-1R antibodies (mAb) based on published sequences for therapeutic mAbs from Pfizer (CP751,871, IgG2 kappa), Amgen (AMG479, IgG1 lambda), Merck (h7C10, cloned to R1507 backbone), Imclone (IMC-A12, IgG1 lambda) and Roche (R1507, IgG1). Methods In this study, sequence information for IGF-1R mAbs was extracted from patents and used to clone and transiently express IgGs (*=re-synthesized) in HEK293F cells. In vitro assays for ligand binding, IGF-1R auto-phosphorylation, IGF-1R downregulation, IR co-downregulation, and affinity analyses (Biacore) were used. In vivo comparison was done in BxPC3 xenograft mouse model. Results All antibodies inhibit IGF-1 binding and signaling at low nanomolar levels. While IMC-A12* and R1507 also prevent IGF-2 binding to IGF-1R and subsequent receptor activation, CP751,871* and h7C10* do not inhibit IGF-2 binding and have only limited impact (55%/30% inhibition) on IGF-2 signaling. Analysis of IGF-1R phosphorylation in 0.5 % FCS medium revealed that all antibodies except R1507 exert agonistic activity. Interestingly, Fab fragments of agonistic mAbs became antagonistic, indicating that bivalent binding is necessary for agonistic effects. All mAb (200nM, 24h treatment of MCF-7 cells) with the exception of AMG479* efficiently downregulated 78–82% the IGF-1R. Analysis of the same cell lysates revealed however striking differences in IR co-downregulation, a mechanism discussed as possible cause of clinical hyperglycemia. R1507 had the least side effects on Insulin co-downregulation (9%) compared to h7C10* (15%), CP751,871* (23%) and IMC-A12* (46%). Differences were also seen in the binding kinetics. Both AMG479* and R1507 showed faster koff rates resulting in shorter retention times at the receptor. Since kon/koff rates are discussed to influence tumor penetration (1), we compared downregulation of IGF-1R by R1507 and CP751,871* in xenograft tumors. Although both mAbs downregulate IGF-1R in vitro to the same extent, R1507 was significantly more effective in the in-vivo setting. Conclusion The head to head comparison of IGF-1R mAbs revealed differences in regard to binding properties, tumor penetration, IR codownregulation, and inhibition of signaling via the ligand IGF-2. Reference List 1. Adams, G. P., Schier, R., McCall, A. M., Simmons, H. H., Horak, E. M., Alpaugh, R. K., Marks, J. D., and Weiner, L. M. (2001) Cancer Res. 61, 4750–4755 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-397. doi:10.1158/1538-7445.AM2011-LB-397

Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H(2) (16)O(2) and H(2) (18)O(2) labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the (18)O/(16)O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor

The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val299–Val307 within the eighth helical domain (α-H8) adjacent to the palmitoylated residues at Cys308–Cys311. However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys297, Arg302 and Lys304 located within α-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important α-H8 of the hIP. This ER export motif, defined by “K/R(X)4K/R(X)K/R,” has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.

The Integrin Co-Activator Kindlin-2 Plays a Critical Role In Angiogenesis and Blood Vessel Integrity

AbstractAbstract 4Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Our previous study demonstrated that reduction of kindlin-2 levels in human endothelial cells results in defective adhesive and migratory responses, suggesting that kindlin-2 may be implicated in angiogenesis. We tested this hypothesis in the kindlin-2+/− mice utilizing murine RM1 prostate tumor and Matrigel implant models. Staining of tumor sections for EC with CD31 showed shorter and thinner blood vessels and reduced vascular area by 3.5-fold in tumors grown in kindlin-2+/− mice compared to WT mice (P=0.0186, n=6). The vessels that did form in the kindlin-2+/− mice were immature as they lacked smooth muscle cells and pericytes, had thinner basement membrane, and were leaky as evidenced by increased by 2-fold area for plasma-derived fibrin in tumor sections (P=0.0006, n=5). Consistent with the blunted angiogenic response and vascular leakiness in the kindlin-2+/− mice, the tumors grown in kindlin-2+/− animals had 2-fold larger necrotic core and were 2.5-fold smaller than those derived in WT mice (P=0.042, n=7). Also, the permeability of preexisting blood vessels in ear and dorsal skin of WT and kindlin-2+/− mice was compared after injection of Evans blue dye. Baseline permeability of vasculature in ear and dorsal skin was enhanced by ∼70–100 % in kindlin-2+/− mice as compared WT mice (P<0.01, n=6). Application of mustard oil, a proinflammatory stimulus, on ear skin or VEGF on dorsal skin, enhanced permeability in WT mice by ∼75–80 % (P<0.05, n=6) while it had a negligible effect in the kindlin-2+/− mice (P=0.159, n=6). The impaired angiogenic response in the kindlin-2+/− mice was confirmed in a Matrigel model where VEGF-induced angiogenesis was abnormal. CD31 staining showed that in contrast to the matrigel plugs derived from WT mice, those obtained from the kindlin-2+/− mice contained shorter and thinner vessels, and the few thicker CD31+ structures that did form in the kindlin-2+/− mice were disorganized. As a consequence of blood leakage from abnormal vessels the Matrigel implants collected from the kindlin-2+/− mice exhibited a 3.5-fold increase in hemoglobin content as compared to WT mice (P=0.0149, n=5).Bone marrow transplant experiments were performed and showed that bone marrow-derived cells did not significantly contribute to abnormal angiogenesis and enhanced vascular permeability observed in the kindlin-2+/− mice. Tumor vascular area, plasma-derived fibrin content, tumor growth and vascular permeability in ear and dorsal skin were similar in WT recipients, which received bone marrow from kindlin-2+/− or WT donors. Aortic endothelial cells isolated from WT and kindlin-2+/− mice exhibited similar expression levels of the β1 and β3 integrins as well as VEGFR2. However, FACS analysis of soluble ligand binding showed significantly reduced activation of the β3 integrins in kindlin-2+/− ECs as compared to WT cells, whereas the β1 integrin activation was similar in both genotypes. Additionally, PMA or VEGF enhanced integrin activation in WT ECs, but failed to do so in the kindlin-2+/− cells. Accordingly, integrin-dependent cellular functions: adhesion, migration and spreading of the EC derived from the kindlin-2+/− mice were significantly blunted on the β3 integrin substrates but not on the β1 substrates. In control experiments, overexpression of kindlin-2 in the kindlin-2+/− EC restored defective spreading on vitronectin while a kindlin-2 that does not bind integrins, did not, indicating specificity. Also, tube formation by kindlin-2+/− ECs was significantly impaired compared to those developed by WT ECs. Taken together, kindlin-2 plays an important role in angiogenesis as well as in maintenance of vascular integrity. Disclosures:No relevant conflicts of interest to declare.

DFT and Ab initio computational study on the reactivity sites of the GABA and its agonists, such as CACA, TACA, DABA, and muscimol: In the gas phase and dielectric media

AbstractThe reactive behavior of GABA and its agonist molecules have been investigated using B3LYP hybrid density functional method at the 6‐311++G** basis set level, in the gas phase and dielectric media. The calculations have been performed to obtain optimized geometries, relative energies, net atomic charges, HOMO, and LUMO energies. Solvent effects have been analyzed using isodensity polarized continuum model (IPCM) for four different solvents, which are chloroform, ethanol, DMSO, and H2O. The results have suggested that both the amino group and carboxyl group have been two of the most important active site for interactions between all compounds and their receptors. In addition they have suggested that the amino group reflects the difference in biological activity for each all molecules. The structures of these agonists have provided an essential foundation for subsequent structure‐activity analysis of ligand binding at GABA receptors, neuronal uptake inhibitors and transporters. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Interaction Kinetic and Structural Dynamic Analysis of Ligand Binding to Acetylcholine-Binding Protein

The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein and acetylcholine, nicotine, epibatidine, and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model, and a structural interpretation of the interaction was obtained by molecular modeling involving induced fit and loop flexibility simulations. The data suggest that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB 69–333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB 69–333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB 69–333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.

Interdependence of Receptor Activation and Ligand Binding in HCN2 Pacemaker Channels

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.

The Kinetics of Cooperative Cofilin Binding Reveals Two States of the Cofilin-Actin Filament

The interaction of cofilin with actin filaments displays positive cooperativity. The equilibrium binding and associated thermodynamic properties of this interaction are well described by a simple, one-dimensional Ising model with nearest neighbor interactions. Here we evaluate the kinetic contributions to cooperative binding and the ability of this model to account for binding across a wide range of cofilin concentrations. A Monte Carlo-based simulation protocol that allows for nearest-neighbor interactions between adjacent binding sites was used to globally fit time courses of human cofilin binding to human nonmuscle (β-, γ-) actin filaments. Several extensions of the one-dimensional Ising model were tested, and a mechanism that includes isomerization of the actin filament was found to best account for time courses of association as well as irreversible dissociation from a saturated filament. This model predicts two equilibrium states of the cofilin-actin, or cofilactin, filament, and the resulting set of binding parameters are in agreement with equilibrium thermodynamic parameters. We conclude that despite its simplicity, this one-dimensional Ising model is a reliable model for analyzing and interpreting the energetics and kinetics of cooperative cofilin-actin filament interactions. The model predicts that severing activity associated with boundaries between bare and decorated segments will not be linear, but display a transient burst at short times on cofilin activation then dissipate due to a kinetic competition between severing activity and cofilin binding. A second peak of severing activity is predicted to arise from irreversible cofilin dissociation on inactivation. These behaviors predict what we believe to be novel mechanisms of cofilin severing and spatial regulation of actin filament turnover in cells. The methods developed for this system are generally applicable to the kinetic analysis of cooperative ligand binding to linear polymers.

Development of novel aryl hydrocarbon receptor antagonists

Increasing evidence indicates that the aryl hydrocarbon receptor (AHR) plays a role in the development and progression of human chronic diseases. Thus, developing appropriate antagonists that target the AHR may not only provide tools to better understand how the AHR signaling pathway modifies human diseases but may ultimately result in novel pharmacological interventions. With this in mind, we have synthesized and characterized a privileged library of putative AHR antagonists that represent derivatives of CH‐223191. Ligand binding analyses have identified at least three derivatives that exhibit equal to or higher ligand binding affinity that the parent compound, CH‐223191. Using reporter assays and mRNA expression analyses, we have found that these CH‐223191 derivatives are not only effective at blocking TCDD‐induced and basal AHR activity, but display minimal agonistic properties. Interestingly, one derivative, AL‐3 appears to synergize with the AHR agonist TCDD with respect to AHR activity. Taken together, these data set the stage for understanding the rules by which optimal AHR antagonists may be developed. This work is supported by ES014849

Structure Dynamics and Allosteric Regulation of the E. Coli High-Affinity Methionine Transporter Metni

The high-affinity uptake of methionine by Escherichia coli is mediated by MetNI, a member of the methionine uptake transporter family of ATP-binding cassette (ABC) transporters. We previously reported the crystal structure of MetNI at 3.7 A resolution and a brief analysis of the methionine mediated trans-inhibition of the transporters' ATPase activity1. Here, we report two new crystal structures of the MetNI transporter, solved at 2.8 and 4.0 A resolution. While both structures of MetNI reveal the transporter adopting an inward-facing conformation, significant changes are observed in the conformation of the MetI transmembrane, MetN nucleotide binding, and MetN-C2 carboxyl-terminal regulatory domains. The conformational changes can be described primarily as rigid-body movements which result in a partial closing of the MetN nucleotide binding domains, and a simultaneous rotational rearrangement of the MetN-C2 regulatory domain. The kinetic properties of trans-inhibition have also been characterized by an analysis of ligand binding on the ATPase activity of wild type and specific MetN-C2 regulatory domain mutants.1. Kadaba N, Kaiser J, Johnson E, Lee A, and Rees D C. The high affinity E. coli methionine ABC transporter: structure and allosteric regulation. Science. 2008 Jul 11;321(5886):250-3.

The Kinetics of Cooperative Cofilin Binding to Actin Filaments

The interaction of cofilin with actin filaments displays positive cooperativity. The equilibrium binding and associated thermodynamic parameters of this interaction are well described by a simplified, one-dimensional Ising model with nearest neighbor interactions [De La Cruz (2005) J. Mol. Biol. 346, 557-564]. Here we evaluate the ability of the model to account for cooperative association kinetics and to determine the kinetic contributions to cooperative binding. A Monte Carlo based simulation protocol that allows for nearest-neighbor interactions between adjacent binding sites was employed to globally fit time courses of cofilin binding. A consistent set of binding parameters in good agreement with the equilibrium thermodynamic parameters describes well the experimental data across a wide range of cofilin concentrations. We conclude that despite its simplicity, the one-dimensional Ising model with nearest neighbor cooperative interactions, is a reliable model for analyzing and interpreting the thermodynamics and kinetics of cooperative cofilin-actin filament interactions. The methods developed for this system will be applicable to the kinetics analysis of cooperative ligand binding to linear biological polymers.

Phytochemicals as Modulators of PPARs and RXRs

Welcome to this special issue of PPAR Research on “Phytochemicals as modulators of PPARs and RXRs.” Plants have played a crucial role in the prevention and treatment of human diseases for thousands of years. A significant number of contemporary drugs with high efficacy are derived from herbal origin. These natural phytochemicals, including certain nutrients, are therefore of great medicinal value. Peroxisome proliferator-activated receptors (PPARs) belong to the steroid hormone receptor superfamily that bind to and are activated by fatty acids, eicosanoids, and a group of xenobiotics called peroxisome proliferators. Each of these receptors binds to specific peroxisome proliferator response elements (PPRE) as a heterodimer with a retinoid X receptor (RXR). Binding to these receptors has been proven to control the pathological conditions associated with obesity, aging-related diseases, inflammation, immune disorder, cell cycle regulation as well as cancer. There is a strong mechanistic basis for PPAR and RXR targeting, as these nuclear receptors are transcriptional factors that modulate gene expression relevant to the control of blood glucose and lipids, as well as the processes of inflammation and carcinogenesis. Since PPARs play an important role in lipid metabolism, the search for natural ligands had begun with fatty acids and eicosanoids. In fact, the discovery that many natural dietary compounds, especially phytochemicals, are PPAR activators is of great significance for human health. With metabolic syndrome, diabetes, and chronic systemic inflammatory diseases now reaching pandemic proportions, the importance of the discovery of natural PPAR ligands is self-evident. The special issue begins with a review of terpenoids as modulators of the PPAR family. To date more than 40,000 terpenoids have been described. Also known as isoprenoids, terpenoids constitute one of the largest and most diverse group of phytochemicals. Members of this phytochemical family are well known to us as the flavours of cinnamon, ginger, and cloves and the scents of camphor and eucalyptus. The vitamin A precursors known as carotenoids and psychoactive cannabinoids are also forms of terpenoid. In this issue, T. Goto and colleagues review the biosynthesis, metabolism, and plant origins of various dietary terpenoids. The physiological effects of these compounds on carbohydrate and lipid metabolism are described in terms of their PPARγ and PPARα activating abilities. This is followed by the research article on the kinetic assessment and therapeutic modulation of metabolic and obesity-associated inflammatory profiles by a high-fat diet, whereas the whole process was found to be mediated by PPAR activation. Another research article has proposed glycyrrhizic acid, the bioactive compound extracted from roots of licorice (Glycyrrhiza glabra), as a potent PPARγ agonist. This phytochemical is capable of ameliorating metabolic syndrome by affecting glucose homeostasis, lipid metabolism, and adipogenesis. The focus is then shifted to the findings on verbascoside, a phytochemical that exerts a strong anti-inflammatory activity against the development of experimental inflammatory bowel disease. This work by the S. Cuzzocrea team has proven that verbascoside is a PPARα ligand by using a knock-out animal model. S. N. Lewis and colleagues conclude this special issue with a comprehensive review of computational approaches to the discovery of phytochemical ligands for PPARα and PPARγ. This timely review is an excellent reference document for the existing 3D structures of PPAR receptors cocrystallized with a variety of ligands and available on-line at the RCSB database. Lewis gives an expert description of the potential for in silico screening of phytochemical databases. The challenges of applying computational techniques to the analysis of a flexible ligand binding to a flexible receptor are described. This review is a most helpful exposition of the state-of-the-art computational methods regarding natural PPAR ligand discovery. Joshua K. Ko Susanna S. Lee Harry Martin

Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch.

The thi-box riboswitch regulates gene expression in response to the intracellular concentration of thiamine pyrophosphate (TPP) in archaea, bacteria, and eukarya. To complement previous biochemical, genetic, and structural studies of this phylogenetically widespread RNA domain, we have characterized its interaction with TPP by isothermal titration calorimetry. This shows that TPP binding is highly dependent on Mg(2+) concentration. The dissociation constant decreases from approximately 200 nM at 0.5 mM Mg(2+) concentration to approximately 9 nM at 2.5 mM Mg(2+) concentration. Binding is enthalpically driven, but the unfavorable entropy of binding decreases as Mg(2+) concentration rises, suggesting that divalent cations serve to pre-organize the RNA. Mutagenesis, biochemical analysis, and a new crystal structure of the riboswitch suggest that a critical element that participates in organizing the riboswitch structure is the tertiary interaction formed between the P3 and L5 regions. This tertiary contact is distant from the TPP binding site, but calorimetric analysis reveals that even subtle mutations in L5 can have readily detectable effects on TPP binding. The thermodynamic signatures of these mutations, namely decreased favorable enthalpy of binding and small effects on entropy of binding, are consistent with the P3-L5 association contributing allosterically to TPP-induced compaction of the RNA.

Diverse vasopressin V2 receptor functionality underlying partial congenital nephrogenic diabetes insipidus

X-linked congenital nephrogenic diabetes insipidus (CNDI) is characterized by a defective renal response to the antidiuretic hormone (AVP) due to variations in the arginine vasopressin receptor 2 (AVPR2) gene. In a unique group of patients, the renal insensitivity to the effects of AVP is incomplete resulting in a partial phenotype. To investigate the molecular defects, two previously published variations in the AVPR2 gene, known to cause a partial CNDI phenotype, were expressed in transiently transfected human embryonic kidney cells. One variation (p.Arg104Cys) is located in the first extracellular loop and the other variation (p.Ser329Arg) is located in the intracellular COOH terminal of the receptor protein. Western blotting showed almost equal amounts of WT-V2R and Arg104Cys-V2R protein at steady state, whereas the level of Ser329Arg-V2R protein was lower. Confocal microscopy established that WT-V2R and Arg104Cys-V2R are localized on the cellular surface while the Ser329Arg-V2R primarily accumulates within the endoplasmic reticulum resulting in reduced surface expression. Ligand binding analysis demonstrated that the B(max) for cells expressing Arg104Cys-V2R and Ser329Arg-V2R were 14.8- and 2.5-fold lower than B(max) for WT-V2R, respectively. AVP affinity (1/K(d)) for WT-V2R and the Ser329Arg-V2R was similar while 1/K(d) for Arg104Cys-V2R was increased. cAMP assay revealed that cells expressing p.Arg104Cys-V2R or p.Ser329Arg-V2R produced 1.7- and 6.8-fold lower amounts of cAMP compared with WT-V2R, respectively. In conclusion, ligand binding and signal transduction capability are dependent on localization of the amino acid variation. Striking divergences at the level of receptor functionality may thus underlie similar clinical phenotypes in CNDI.

Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC50 value for the dipeptide Ala-Ala was measured to 22mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC50 values of 0.3mM and 1.5mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil ( Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.