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1.  Immunosuppressive peptides and their therapeutic applications☆ 
Drug Discovery Today  2014;19(5):645-653.
Highlights
•T cell signaling has a pivotal role in autoimmunity and immunosuppression.•Immunosuppressive pharmaceuticals often exhibit severe side-effects in patients.•Gene-encoded peptides have potential as immunosuppressive drug candidates.•Cyclotides are stable peptides that offer enhanced oral administration properties.
The immune system is vital for detecting and evading endogenous and exogenous threats to the body. Failure to regulate this homeostasis leads to autoimmunity, which is often associated with malfunctioning T cell signaling. Several medications are available to suppress over-reactive T lymphocytes, but many of the currently marketed drugs produce severe and life-threatening side-effects. Ribosomally synthesized peptides are gaining recognition from the pharmaceutical industry for their enhanced selectivity and decreased toxicity compared with small molecules; in particular, circular peptides exhibit remarkable stability and increased oral administration properties. For example, plant cyclotides effectively inhibit T lymphocyte proliferation. They are composed of a head-to-tail cyclized backbone and a cystine-knot motif, which confers them with remarkable stability, thus making them attractive pharmaceutical tools.
doi:10.1016/j.drudis.2013.12.002
PMCID: PMC4042018  PMID: 24333193
2.  From ethnopharmacology to drug design 
Nature’s diversity is one of the biggest resources of therapeutic lead compounds. Traditionally-used herbal remedies harbor a variety of bioactive compounds providing researchers with starting points for drug development. Ethnopharmacological investigations of uterotonic plant preparations identified a class of circular and disulfide-rich peptides, called cyclotides, to exhibit strong uterine contractions. In humans one of the physiological regulators of the myometrial contractility is the nonapeptide oxytocin acting on its cognate G protein-coupled receptors. They are considered to represent one of the most promising drug targets with ~30% of all currently marketed drugs acting on these transmembrane receptors. Based on observed similarities regarding the activity and structure of plant cyclotides with human oxytocin we analyzed the pharmacological principle of their action and identified the oxytocin and vasopressin 1a receptors as molecular targets of cyclotides from the Rubiaceae plant Oldenlandia affinis. Using a synthetic approach, the sequence of the native cyclotide kalata B7 was used to design oxytocin-like nonapeptides with nanomolar affinity and selectivity for the oxytocin receptor. This provides formal proof for the use of cyclotides as templates in peptide ligand design. At a more general level, mining of naturally-occurring peptides is a promising tool for the identification and the design of novel G protein-coupled receptor ligands.
doi:10.4161/cib.27583
PMCID: PMC3925455  PMID: 24567782
cyclotides; oxytocin; G protein-coupled receptors; peptide; plant; ligand; selectivity
3.  Cyclotide Discovery in Gentianales Revisited—Identification and Characterization of Cyclic Cystine-Knot Peptides and Their Phylogenetic Distribution in Rubiaceae Plants 
Biopolymers  2013;100(5):10.1002/bip.22328.
Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ~300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.
doi:10.1002/bip.22328
PMCID: PMC3816352  PMID: 23897543
cyclotides; peptidomics; transcriptomics; ipecac; Rubiaceae; Carapichea; Notopleura; Psychotrieae; Palicoureeae
4.  Physiology of invertebrate oxytocin and vasopressin neuropeptides 
Experimental physiology  2013;99(1):10.1113/expphysiol.2013.072561.
Neuropeptides and regulatory peptide hormones control many developmental, physiological and behavioural processes in animals, including humans. The nonapeptides oxytocin and arginine vasopressin are produced and released by the pituitary gland and have actions on many organs and tissues. Receptive cells possess particular receptors to which the peptides bind as ligands, leading to activation of G-protein-coupled receptors, hence cellular responses. In humans and other mammalian species, oxytocin and vasopressin mediate a range of peripheral and central physiological functions that are important for osmoregulation, reproduction, complex social behaviours, memory and learning. The origin of the oxytocin/vasopressin signalling system is thought to date back more than 600 million years. All vertebrate oxytocin- and vasopressin-like peptides have presumably evolved from the ancestral nonapeptide vasotocin by gene duplication and today are present in vertebrates, including mammals, birds, reptiles, amphibians and fish. Oxytocin- and vasopressin-like peptides have been identified in several invertebrate species, including molluscs, annelids, nematodes and arthropods. Members of this peptide family share high sequence similarity, and it is possible that they are functionally related across the entire animal kingdom. However, it is evident that not all animals express oxytocin/vasopressin neuropeptides and that there is little information available about the biology and physiology of this signalling system of invertebrates and, in particular, of insects, which represent more than half of all known living organisms. This report describes the discovery of novel oxytocin- and vasopressin-like peptides in arthropods and summarizes the status quo of the functional relevance of this neuropeptide signalling system in invertebrates, which will have beneficial implications for the design of selective and potent ligands to human oxytocin and vasopressin receptors.
doi:10.1113/expphysiol.2013.072561
PMCID: PMC3883647  PMID: 23955310
5.  Exploring bioactive peptides from natural sources for oxytocin and vasopressin drug discovery 
Future medicinal chemistry  2012;4(14):10.4155/fmc.12.108.
doi:10.4155/fmc.12.108
PMCID: PMC3816348  PMID: 23043476
ant; cone snail; conopressin; G-protein-coupled receptor; ligand-based drug design; oxytocin; selectivity issue; vasopressin; vasotocin
6.  Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine 
Journal of Ethnopharmacology  2012;143(1):377-382.
Ethnopharmacological relevance
Pregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation.
Aim of study
Obtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level.
Materials and methods
Semi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model.
Result
The plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure.
Conclusion
The folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.
Graphical abstract
Pregnant woman in Nigeria rely on traditional herbal medicine to induce or ease labor, and to treat childbirth-related complications. Nine plant species have been documented and characterized for their uterotonic properties.
doi:10.1016/j.jep.2012.06.042
PMCID: PMC3430860  PMID: 22766472
UM, use-mentions; hTERT-HM, human uterine myometrial smooth muscle cells; Maternal healthcare; Uterus contractility; Labor; Postpartum care
7.  Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature 
Natural product reports  2013;30(1):108-160.
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
doi:10.1039/c2np20085f
PMCID: PMC3954855  PMID: 23165928
8.  Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism 
PLoS ONE  2013;8(6):e68016.
Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.
doi:10.1371/journal.pone.0068016
PMCID: PMC3694003  PMID: 23840803
cyclotide; lymphocytes; proliferation; immunosuppression; plant; peptide; cytokine
9.  Immunosuppressive activity of an aqueous Viola tricolor herbal extract 
Journal of ethnopharmacology  2013;151(1):299-306.
Ethnopharmacological relevance
Heartsease (Viola tricolor L.), a member of the Violaceae family, has a long history as a medicinal plant and has been documented in the Pharmacopoeia of Europe. Due to its anti-inflammatory properties it is regarded as a traditional remedy against skin diseases, for example for the treatment of scabs, itching, ulcers, eczema or psoriasis, and it is also used in the treatment of inflammation of the lungs and chest such as bronchitis or asthma. Because T-cells play an important role in the pathological process of inflammatory diseases we investigated the effect of an aqueous Viola extract on lymphocyte functions and explored the ‘active’ principle of the extract using bioactivity-guided fractionation.
Material and Methods
An aqueous Viola extract was prepared by C18 solid-phase extraction. Effects on proliferation of activated lymphocytes (using the cell membrane permeable fluorescein dye CFSE), apoptosis and necrosis (using annexin V and propidium iodide staining), interleukin-2 (IL-2) receptor expression (using fluorochrome-conjugated antibodies) and IL-2 cytokine secretion (using an ELISA-based bead array system) were measured by flow cytometry. Influence on lymphocyte polyfunctionality was characterized by Viola extract-induced production of IFN-γ and TNF-α, as well as its influence on lymphocyte degranulation activity. Fractionation and phytochemical analysis of the extract were performed by RP-HPLC and mass spectrometry.
Results
The aqueous Viola extract inhibited proliferation of activated lymphocytes by reducing IL-2 cytokine secretion without affecting IL-2 receptor expression. Similarly, effector functions were affected as indicated by the reduction of IFN-γ and TNF-α production; degranulation capacity of activated lymphocytes remained unaffected. Bioassay-guided fractionation and phytochemical analysis of the extract led to identification of circular plant peptides, so called cyclotides, as bioactive components.
Conclusion
An aqueous Viola extract contains bioactive cyclotides, which inhibit proliferation of activated lymphocytes in an IL-2 dependent manner. The findings provide a rationale for use of herbal Viola preparations in the therapy of disorders related to an overactive immune system. However, further studies to evaluate its clinical potency and potential risks have to be performed.
doi:10.1016/j.jep.2013.10.044
PMCID: PMC3918579  PMID: 24216163
Violaceae; Viola tricolor L.; Immunosuppression; Cyclotides; Psoriasis; Anthroposophical medicine; Phytotherapy
10.  Insights into the molecular evolution of oxytocin receptor ligand binding 
Biochemical Society Transactions  2013;41(Pt 1):197-204.
The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OT's interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.
doi:10.1042/BST20120256
PMCID: PMC3634130  PMID: 23356283
arginine vasopressin; binding; GPCR (G-protein-coupled receptor); homology model; OT (oxytocin); vasotocin; AVP, arginine vasopressin; CTR, cephalotocin receptor; GPCR, G-protein-coupled receptor; ICL, intracellular loop; OT, oxytocin; OTR, oxytocin receptor; TM, transmembrane; VT, vasotocin
13.  Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry 
Amino Acids  2012;44(2):581-595.
Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-012-1376-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00726-012-1376-x
PMCID: PMC3549257  PMID: 22890611
Viola ignobilis; Circular; Cystine-knot; Oxidative folding; Vigno; Peptidomics
14.  Uterotonic Plants and their Bioactive Constituents 
Planta medica  2010;77(3):207-220.
Abnormalities in the process of uterine muscle contractility during pregnancy and birth can have major clinical implications, including preterm labour, which is the single largest cause of maternal and prenatal mortality in the Western world and a major contributor to childhood developmental problems. In contrast, induction of labour may be necessary in certain conditions. Currently used interventional therapies to suppress (tocolytic agents) or to induce (uterotonic agents) uterine contractions lack potency and/or selectivity and can have harmful side effects for mother and baby. Nature’s diversity has always been, and still is, one of the biggest resources of therapeutic lead compounds. Many natural products exhibit biological activity against unrelated targets, thus providing researchers with starting points for drug development. In this review we will provide an overview of uterine muscle physiology, describe currently available biological screening procedures for testing of uterotonic plant compounds and will summarise traditionally-used uterotonic plants, their active components and their mechanisms, primarily focusing on uterotonic active circular plant peptides called cyclotides. Finally we will comment on the discovery of novel cyclotide-producing plant species and the possibility for the development of novel plant-derived uterotonic and tocolytic drugs.
doi:10.1055/s-0030-1250317
PMCID: PMC3407953  PMID: 20845261
women’s health; gynaecology; labour; myometrial smooth muscle; oxytocin; cyclotides; plants
15.  Do Plant Cyclotides Have Potential As Immunosuppressant Peptides? 
Journal of natural products  2012;75(2):167-174.
Cyclotides are an abundant and diverse group of ribosomally synthesized plant peptides containing a cyclic cystine-knotted structure that confers them with remarkable stability. They are explored for their distribution in plants, although little is known about the individual peptide content of a single species. Therefore, we chemically analyzed the crude extract of the coffee-family plant Oldenlandia affinis using a rapid peptidomics workflow utilizing nano-LC-MS, peptide reconstruct with database identification, and MS/MS automated sequence analysis to determine its cyclotide content. Biologically, cyclotides are mainly explored for applications in agriculture and drug design; here we report their growth-inhibiting effects on primary cells of the human immune system using biological and immunological end points in cell-based test systems. LC-MS quantification of the active O. affinis plant extract triggered the characterization of the antiproliferative activity of kalata B1, one of the most abundant cyclotides in this extract, on primary activated human lymphocytes. The effect has a defined concentration range and was not due to cytotoxicity, thus opening a new avenue to utilize native and synthetically optimized plant cyclotides for applications in immune-related disorders and as immunosuppressant peptides.
doi:10.1021/np200722w
PMCID: PMC3399773  PMID: 22272797
16.  Discovery of Defense- and Neuropeptides in Social Ants by Genome-Mining 
PLoS ONE  2012;7(3):e32559.
Natural peptides of great number and diversity occur in all organisms, but analyzing their peptidome is often difficult. With natural product drug discovery in mind, we devised a genome-mining approach to identify defense- and neuropeptides in the genomes of social ants from Atta cephalotes (leaf-cutter ant), Camponotus floridanus (carpenter ant) and Harpegnathos saltator (basal genus). Numerous peptide-encoding genes of defense peptides, in particular defensins, and neuropeptides or regulatory peptide hormones, such as allatostatins and tachykinins, were identified and analyzed. Most interestingly we annotated genes that encode oxytocin/vasopressin-related peptides (inotocins) and their putative receptors. This is the first piece of evidence for the existence of this nonapeptide hormone system in ants (Formicidae) and supports recent findings in Tribolium castaneum (red flour beetle) and Nasonia vitripennis (parasitoid wasp), and therefore its confinement to some basal holometabolous insects. By contrast, the absence of the inotocin hormone system in Apis mellifera (honeybee), another closely-related member of the eusocial Hymenoptera clade, establishes the basis for future studies on the molecular evolution and physiological function of oxytocin/vasopressin-related peptides (vasotocin nonapeptide family) and their receptors in social insects. Particularly the identification of ant inotocin and defensin peptide sequences will provide a basis for future pharmacological characterization in the quest for potent and selective lead compounds of therapeutic value.
doi:10.1371/journal.pone.0032559
PMCID: PMC3308954  PMID: 22448224
17.  Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor* 
The Journal of Biological Chemistry  2013;288(40):28831-28844.
Background: The A2A receptor is known to accumulate in the endoplasmic reticulum.
Results: Mass spectrometry identified molecular chaperones (HSP90 and HSP70) bound to the A2A receptor.
Conclusion: Sequential recruitment of chaperones to the cytosolic face of the A2A receptor is consistent with a heat-shock protein relay assisting folding.
Significance: The observations are consistent with a chaperone/COPII exchange model, where heat-shock proteins bound to the receptor preclude its premature ER export.
The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.
doi:10.1074/jbc.M113.464776
PMCID: PMC3789979  PMID: 23965991
7-Helix Receptor; Adenosine Receptor; Adenylate Cyclase (Adenylyl Cyclase); Endoplasmic Reticulum (ER); G Protein-coupled Receptors (GPCR); G Proteins; Heat-Shock Protein
18.  Proteome analysis of the hyaluronic acid-producing bacterium, Streptococcus zooepidemicus 
Proteome Science  2009;7:13.
Background
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a commensal of horses and an opportunistic pathogen in many animals and humans. Some strains produce copious amounts of hyaluronic acid, making S. zooepidemicus an important industrial microorganism for the production of this valuable biopolymer used in the pharmaceutical and cosmetic industry. Encapsulation by hyaluronic acid is considered an important virulence factor in other streptococci, though the importance in S. zooepidemicus remains poorly understood. Proteomics may provide a better understanding of virulence factors in S. zooepidemicus, facilitate the design of better diagnostics and treatments, and guide engineering of superior production strains.
Results
Using hyaluronidase to remove the capsule and by optimising cellular lysis, a reference map for S. zooepidemicus was completed. This protocol significantly increased protein recovery, allowing for visualisation of 682 spots and the identification of 86 proteins using mass spectrometry (LC-ESI-MS/MS and MALDI-TOF/TOF); of which 16 were membrane proteins.
Conclusion
The data presented constitute the first reference map for S. zooepidemicus and provide new information on the identity and characteristics of the more abundantly expressed proteins.
doi:10.1186/1477-5956-7-13
PMCID: PMC2670282  PMID: 19327162
19.  Reengineering the Collision Coupling and Diffusion Mode of the A2A-adenosine Receptor 
The Journal of Biological Chemistry  2012;287(50):42104-42118.
Background: The A2A receptor engages Gs by restricted collision coupling and lacks a palmitoyl moiety in its C terminus.
Results: Engineering palmitoylated cysteine into the C terminus relieved restricted collision coupling and resulted in accelerated diffusion of the agonist-liganded A2A receptor.
Conclusion: Restricted collision coupling arises from limits imposed on receptor diffusion.
Significance: Agonist induced confinement of the A2A receptor in a structure consistent with a lipid raft.
The A2A-adenosine receptor undergoes restricted collision coupling with its cognate G protein Gs and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A2A-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A2A receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A2A-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A2A receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A2A-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A2A receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.
doi:10.1074/jbc.M112.393579
PMCID: PMC3516756  PMID: 23071116
Adenosine Receptor; Adenylate Cyclase; Cyclic AMP (cAMP); G Protein-coupled Receptors (GPCR); G Proteins
21.  The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters* 
The Journal of Biological Chemistry  2012;287(22):18524-18534.
Background: Ibogaine is a noncompetitive inhibitor of SERT that stabilizes the transporter in an inward-open conformation.
Results: Ibogaine binds to a site accessible from the cell exterior that does not overlap with the substrate-binding site.
Conclusion: Ibogaine binds to a novel binding site on SERT and DAT.
Significance: This study provides a mechanistic understanding of an unique inhibitor of SERT and DAT.
Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.
doi:10.1074/jbc.M112.343681
PMCID: PMC3365767  PMID: 22451652
Addiction; Dopamine Transporters; Drug Action; Electrophysiology; Serotonin Transporters
22.  Physiology of invertebrate oxytocin and vasopressin neuropeptides 
Experimental Physiology  2013;99(1):55-61.
New findingsWhat is the topic of this review?This article describes the discovery and function of invertebrate oxytocin and vasopressin neuropeptides.What advances does it highlight?The novel discovery of oxytocin-like peptides in arthropods is described. An up-to date overview is gven of the functional role (physiology and behaviour) of oxytocin and vasopressin signalling. The application of natural peptides for drug development is discussed.
Neuropeptides and regulatory peptide hormones control many developmental, physiological and behavioural processes in animals, including humans. The nonapeptides oxytocin and arginine vasopressin are produced and released by the pituitary gland and have actions on many organs and tissues. Receptive cells possess particular receptors to which the peptides bind as ligands, leading to activation of G-protein-coupled receptors, hence cellular responses. In humans and other mammalian species, oxytocin and vasopressin mediate a range of peripheral and central physiological functions that are important for osmoregulation, reproduction, complex social behaviours, memory and learning. The origin of the oxytocin/vasopressin signalling system is thought to date back more than 600 million years. All vertebrate oxytocin- and vasopressin-like peptides have presumably evolved from the ancestral nonapeptide vasotocin by gene duplication and today are present in vertebrates, including mammals, birds, reptiles, amphibians and fish. Oxytocin- and vasopressin-like peptides have been identified in several invertebrate species, including molluscs, annelids, nematodes and arthropods. Members of this peptide family share high sequence similarity, and it is possible that they are functionally related across the entire animal kingdom. However, it is evident that not all animals express oxytocin/vasopressin neuropeptides and that there is little information available about the biology and physiology of this signalling system of invertebrates and, in particular, of insects, which represent more than half of all known living organisms. This report describes the discovery of novel oxytocin- and vasopressin-like peptides in arthropods and summarizes the status quo of the functional relevance of this neuropeptide signalling system in invertebrates, which will have beneficial implications for the design of selective and potent ligands to human oxytocin and vasopressin receptors.
doi:10.1113/expphysiol.2013.072561
PMCID: PMC3883647  PMID: 23955310
23.  Molecular Grafting onto a Stable Framework Yields Novel Cyclic Peptides for the Treatment of Multiple Sclerosis 
ACS Chemical Biology  2013;9(1):156-163.
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and is characterized by the destruction of myelin and axons leading to progressive disability. Peptide epitopes from CNS proteins, such as myelin oligodendrocyte glycoprotein (MOG), possess promising immunoregulatory potential for treating MS; however, their instability and poor bioavailability is a major impediment for their use clinically. To overcome this problem, we used molecular grafting to incorporate peptide sequences from the MOG35–55 epitope onto a cyclotide, which is a macrocyclic peptide scaffold that has been shown to be intrinsically stable. Using this approach, we designed novel cyclic peptides that retained the structure and stability of the parent scaffold. One of the grafted peptides, MOG3, displayed potent ability to prevent disease development in a mouse model of MS. These results demonstrate the potential of bioengineered cyclic peptides for the treatment of MS.
doi:10.1021/cb400548s
PMCID: PMC3898541  PMID: 24147816

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