Peptide-binding Receptors Research Articles

HP-01-003 Expression of genes encoding for endopeptidase enzymes (EnPEs) and peptide binding receptors in the human vagina # Comparison to penile erectile tissue

HP-01-003 Expression of genes encoding for endopeptidase enzymes (EnPEs) and peptide binding receptors in the human vagina # Comparison to penile erectile tissue

Genomics, GPCRs and new targets for the control of insect pests and vectors.

The pressing need for new pest control products with novel modes of action has spawned interest in small molecules and peptides targeting arthropod GPCRs. Genome sequence data and tools for reverse genetics have enabled the prediction and characterization of GPCRs from many invertebrates. We review recent work to identify, characterize and de-orphanize arthropod GPCRs, with a focus on studies that reveal exciting new functional roles for these receptors, including the regulation of metabolic resistance. We explore the potential for insecticides targeting Class A biogenic amine-binding and peptide-binding receptors, and consider the innovation required to generate pest-selective leads for development, within the context of new PCR-targeting products to control arthropod vectors of disease.

Biological Effects Induced by 68Ga-Conjugated Peptides in Human and Rodent Tumor Cell Lines

Positron emission tomography (PET) with 68Ga-labeled peptides is a promising option in the imaging of tumors expressing peptide-binding receptors. PET scanning is non-invasive but involves internal exposure to ionizing radiation which could subsequently generate biological effects, particularly on targeted cells. The aim of the present study was to investigate the potential biological effects of 68Ga-labeled peptides on different targeted tumor cells in vitro. Human U87MG and HT-29 cells and murine AR42J and B16F10 cells were incubated with the following 68Ga-labeled peptides: 68Ga-NODAGA-c(RGDfK), 68Ga-DOTA-c(RGDfK), 68Ga-NOTA-TATE, 68Ga-DOTA-NOC, 68Ga-NODAGA-NOC, or 68Ga-NOTA-(Cys-39)-Exendin-4. Cell cycle analysis and annexin V/PI assay for apoptosis were performed by flow cytometry. Oxidative stress was evaluated by spectrophotometric measuring the level of reactive oxygen species (ROS) and total thiols. After incubation with 37 kBq/ml (1 µCi/ml) 68Ga-labeled peptides, generally observed an increase of apoptosis and cell cycle arrest, especially in G2/M. Furthermore, the increased ROS generation was mostly correlated with apoptosis. Our data demonstrated the ability of 68Ga-labeled peptides to exert significant inhibitory effects on tumor cell lines, through the cycle dysregulation and induction of apoptosis.

Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog.

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.

Characterization of Ligand Binding to GPCRs Through Computational Methods.

The recent increase in available G protein-coupled receptor structures now contributes decisively to the structure-based ligand design. In this context, computational approaches in combination with medicinal chemistry and pharmacology are extremely helpful. Here, we provide an update on our structure-based computational protocols, used to answer key questions related to GPCR-ligand binding. All combined, these techniques can shed light on ligand binding modes, determine the molecular basis of conformational selection, for agonists and antagonists, as well asof subtype selectivity. To illustrate each of these questions, we will consider examples from existing projects on three families of class A (rhodopsin-like) GPCRs: one small-molecule (nucleotide-like) family, i.e., the adenosine receptors, and two peptide-binding receptors: neuropeptide-Y and angiotensin II receptors. The successful application of the same computational protocols to investigate this diverse group of receptor families gives an idea of the general applicability of our methodology in the characterization of GPCR-ligand binding.

Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus

Thermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication.

Families of allatoregulator sequences: a 2011 perspective1This review is part of a virtual symposium on recent advances in understanding a variety of complex regulatory processes in insect physiology and endocrinology, including development, metabolism, cold hardiness, food intake and digestion, and diuresis, through the use of omics technologies in the postgenomic era.

Three different peptide families have been named “allatostatins” (ASTs), based on their initial purifications which were based on their ability to inhibit juvenile hormone (JH) biosynthesis. These include (i) a family of peptides that have a consensus C-terminal sequence Y/FXFGL-NH2; (ii) a family of peptides with a conserved C-terminal sequence W(X)6W-NH2; and(iii) a family of peptides with C-terminal sequence PISCF, some of which are C-terminally-amidated. Each allatostatin family has functions distinct and apart from the inhibition of JH biosynthesis. A peptide family known as the “allatotropins” serve to stimulate JH biosynthesis. This family of peptides also has been proven to exert multiple effects dependent on the species in question. Genome and peptidome projects are uncovering new members of these families and it is clear that these structures are not just confined to Insecta but are found in a range of invertebrates. The receptors for these neuropeptides have been identified and tested experimentally for specific ligand binding. The Y/FXFGLa-ASTs exert their action through galanin-like receptors, W(X)6Wa-ASTs through a sex peptide-binding receptor, and PISCF-ASTs through somatostatin-like receptors. These receptors are conserved through evolutionary time and are being identified in numerous invertebrates by way of genome projects.

Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30-50%). Closer scrutiny, however, shows that only a modest fraction of (≈60) GPCRs are, in fact, exploited as drug targets, only ≈20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the "orthosteric site"). These additional sites include (i) binding sites for ligands (referred to as "allosteric ligands") that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points.

Abstract 661: Identification of the novel tissue biomarker for breast cancer cells ZR-75-1 using combination of bacterial display and modified affinity chromatography methods

Abstract Identification of the novel tissue biomarker for breast cancer cells ZR-75-1 using combination of bacterial display and modified affinity chromatography methods Introduction: Recognition of biomarkers for cancers is very important for the diagnosis, therapy and prognosis of these kinds of diseases. The study of identifying those novel cancer biomarkers is difficult although people have already tried lots of methods, the numbers and types of effective biomarkers could not meet the needs of medical practices yet. Setting up new methods and identifying novel biomarkers for cancers are working aims for many researchers. Breast cancer is one of the most malignant and common cancer types for women. Even some biomarkers have been identified and prove to play a role in the diagnosis and treatment of cancers, such as estrogen and HER-2, but they cannot be applied to all breast cancer types. The purpose of this work is to combine the bacterial display and modified affinity chromatography methods to identify novel biomarkers for breast cancer cells ZR-75-1. Methods: 1. Bacteria display methods have been used to identify the specific binding peptides for ZR-75-1 cells; 2. Bacteria which express specific binding peptides have been used as the matrix for affinity chromatography; 3. The membrane fraction of cells has been extracted and biotinylated cell membrane lysates have been used to bait the peptide binding receptors; 4. Protein identification has been performed using Nano-LC followed by mass spectrometry; 5. Newly identified membrane receptors for specific peptides need to be justified as novel biomarkers for this kind of cancer by checking the protein membrane localization, interactive proteins and affected possible signaling pathway. Summary and conclusion: Peptides which can interact with breast cancer ZR-75-1 cells have been identified and published in the lab previous paper, but the targeted receptor groups for those peptides have not been established. Glycolytic enzyme aldolase is supposed to be the cytosolic protein which is involved in the cell glycolysis procedure, but our experiment proved that it could be the binding partner for the one of the most specific peptides published before by modified affinity chromatography method. Furthermore, it has also been proved that both aldolase and calveolin-1 can co-locate on the membrane of these non-muscle cells. Therefore, we can predict that this over-expressed membrane localized glycolytic enzyme may be involved in some signal transduction pathways which can initiate tumorigenesis or proliferation besides its original glycolysis activity. Meanwhile, combination of identification of specific binding peptides using bacteria display with modified affinity chromatography methods could be used as the novel methods to identify the tissue biomarkers for cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 661.

A Peptide-binding Receptor Based on Resorc[4]arene and Peptide-Metal Complexes

consists of two different parts: A benzene-lined hydrophobic cleft to which the flexible arms are attached, and the arms based on peptide- metal complexes derived from amino acids. This molecule has a potential substrate-binding cavity, having a benzene-lined hy-drophobic surface with a periphery of hydrogen bond donor/ acceptors. Thus, this molecule might be expected to be capable of interacting selectively with peptide substrates through hy-drophobic interactions and hydrogen bonding. In particular, in this receptor-like molecule, metal ions act as sensitive probes for binding with substrates and providing additional interactions with peptide substrates. Receptor

The amphioxus (Branchiostoma floridae) genome contains a highly diversified set of G protein-coupled receptors

BackgroundG protein-coupled receptors (GPCRs) are one of the largest families of genes in mammals. Branchiostoma floridae (amphioxus) is one of the species most closely related species to vertebrates.ResultsMining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Surprisingly, the Adhesion GPCR repertoire in amphioxus includes receptors with many new domains not previously observed in this family. We found many Rhodopsin GPCRs from all main groups including many amine and peptide binding receptors and several previously uncharacterized expansions were also identified. This genome has however no genes coding for bitter taste receptors (TAS2), the sweet and umami (TAS1), pheromone (VR1 or VR2) or mammalian olfactory receptors.ConclusionThe amphioxus genome is remarkably rich in various GPCR subtypes while the main GPCR groups known to sense exogenous substances (such as Taste 2, mammalian olfactory, nematode chemosensory, gustatory, vomeronasal and odorant receptors) in other bilateral species are absent.

Sequence‐Selective Peptide‐Binding Metallomacrocycles.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Enzyme-based visualization of receptor–ligand binding in tissues

New methods of elucidating the ligand-binding activity of receptors could improve our understanding of receptor function, key events they control, and their presence in normal and pathological states. We describe a method for visualizing receptor–ligand binding in cells and tissues that substitutes fluorescein for radioactive labels, and detects receptor bound, fluoresceinated ligand with an antifluorescein/horseradish peroxidase amplification system. Receptor-bound ligand is then visualized by light microscopy against a standard hemotoxylin-stained background of cell structure. Quantitative versions of the assay provide an apparent dissociation constant and number of receptors per cell at saturation in cell or tissue specimens. Receptors examined include the folate receptor, bombesin peptide-binding receptors, the epidermal growth factor receptor, the neuropeptide Y receptor, the asialoglycoprotein receptor, and RGD peptide-binding integrins. Using fluoresceinated versions of molecules, we show the method can visualize and quantitate receptor-bound ligands in cell culture monolayers and animal tissue specimens. Ligand binding to receptors present in tissues was visualized in normal and pathological samples of human tissue microarrays. The enzyme-amplified detection of receptor-bound fluoresceinated ligand is a simple and nonradioactive-based method that provides information on the receptor activity in tissue specimens.

Sequence-selective Peptide-binding Metallomacrocycles

Since the pioneering work of Pederson, Cram and Lehn, many molecular receptors capable of interacting selectively with various substrates have been described. Particularly, the development of peptide-binding receptors is of great interest because of its relevance to peptide-protein recognition processes in biological systems. Recently, self-assembly by exploiting noncovalent interactions such as metal-ligand coordinate bond is emerging as a novel strategy in construction of peptide-binding synthetic molecules. Here, as the continuing efforts to develop selective peptide-binding receptors, novel C2-symmetric metallomacrocycles are described. Syntheses of receptors 1-4 began with the preparation of the flexible ligand (9), as shown in Scheme 1. DIC-promoted amide coupling reaction between N-Boc-(L)-phenylalanine and 4,4'-methylenedianiline provided the starting material 5. DMAP-catalyzed amide coupling reaction between Boc-deprotected bis-amine of 5 and bis-pentafluorophenyl ester 6, and the subsequent deprotection of allyl groups and imine formation with benzyl amine provided the ligand 9. Metallomacrocycles (1-4) were prepared by mixing ligand 9 and the corresponding metal chloride, acetate or acetoacetonate in ethanol, stirring for 12 hrs under reflux conditions with 55, 52, 49 and 45%, respectively. The products, metal complexes (1-4) are air-stable and moisture-insensitive, and the structures of 1-4 were established by mass spectrum, H NMR spectroscopy, IR and UV spectroscopy. Recently, combinatorial chemistry has become a major tool in the elucidation of the binding properties of receptors. Receptor 2 has the distinct red color due to transition metal ion (Fe(III)), and thus ideal for solid phase color binding assay using encoded combinatorial library of peptide substrates. Receptor 2 was screened against a tripeptide library on hydrophobic polystyrene in CHCl3. 7 The library was prepared by encoded split synthesis and has the general structure Ac-AA3-AA2-AA1-NH(CH2)6-C(O)NH-Polystyrene. Decoding the tripeptides on the colored beads by using electron capture gas chromatography revealed selective peptides-binding properties of receptor (2). The most tightly binding substrates with macrocyclic compounds (2) are shown in Table 1.

Site-specific Disulfide Capture of Agonist and Antagonist Peptides on the C5a Receptor

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.

A Tweezer‐Like Peptide‐Binding Receptor

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A Tweezer-like Peptide-binding Receptor

In recent years, significant progress has been made in the construction of synthetic molecules that selectively bind peptide substrates and other small molecules. Some such molecules have the complex, cage-like structures, but a different and simple structural motif such as molecular tweezer with two substrate binding arms has emerged as having selective peptide-binding properties. Among those are two-armed receptors based on cyclic oligomers of 1,2diamine and isophthalic acid. Although such receptors showed remarkable binding properties, to have further possible applications its binding properties had to be improved. Here, a novel two-armed tweezer-like peptidebinding receptor is described. Receptor (1) consist of two different parts: A linker to which the tweezer arms are attached; and two substrate binding arms based on cyclic tetramer of two isophthalic acids and two 1,2-diaminocyclohexane. Particularly, linker in receptor (1) has transition metal ion (Ru). In this receptor-like molecule, metal ion acts to make potential substrate-binding sites to be preorganized for the effective complexation with suitable substrates. Furthermore, metal ion such as Ru can act as the sensitive probes for binding with substrates and provide the additional interactions between linkers and peptide substrates. Synthesis of 1 began with the preparation of monocyclic intermediates of 1,2-diaminocyclohexanes and isophthalic acid derivatives by the known procedures for the related molecules. Ester bond formation reaction between bis(pentafluorophenyl)ester (2) and 4,4'-hydroxymethyl-2,2'dipyridine (3), and the subsequent reaction with Ru(bpy)2Cl2 in refluxing EtOH provided 1 with 48.2% yield. Recently, combinatorial chemistry has become a major tool in the elucidation of the binding properties of receptors. By using such methods it has become possible to find the binding properties of receptors efficiently, and furthermore to detect subtle differences in receptor-substrate binding that could not have been studied by conventional experiments. Receptor 1 has the distinct red color due to transition metal ion (Ru), and thus ideal for solid phase color binding assay using encoded combinatorial library of peptide substrates. Receptor 1 was screened against a tripeptide library on Scheme 1. Two-armed Tweezerlike Receptor (1).

Cell Adhesion Receptors for Native and Denatured Type I Collagens and Fibronectin in Rabbit Arterial Smooth Muscle Cells in Culture

Cell adhesion molecules serve as specific cell surface receptors for extracellular matrices and contribute to the attachment, spreading, proliferation, and differentiation of vascular cells. We examined the cell adhesion receptors and binding sites on native type I collagen, heat-denatured type 1 collagen, and fibronectin in rabbit arterial smooth muscle cells (SMC) in culture. On fibronectin, anti-α3β1 and anti-α5β1 integrin antibodies and the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) significantly inhibited the attachment and spreading of rabbit SMC after 1 and 24 h of culture, while anti-α1β1 inhibited attachment and spreading only after 1 h. In contrast, the attachment and spreading of the cells on native type I collagen were mediated by α1β1 integrin and the cell-binding sequence which did not contain RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala) after both 1 and 24 h. On heat-denatured type I collagen, α2β1 integrin mediated the cell attachment and spreading after 1 and 24 h and DGEA served as a recognition site for the α2β1 integrin. α1β1 and α3β1 integrins affected only the initial adherence (1 h after plating) of the cells to denatured type I collagen. These findings suggest that rabbit SMC in culture can recognize the native and unfolded triple helical structures of type I collagen by interacting with the collagen fibril-binding receptor (α1β1 integrin) and collagen peptide-binding receptors (α2β1 and α3β1 integrins). Moreover, α1β1 integrin may mediate the initial adherence to each substrate.