A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Modulating receptor function through RAMPs:can they represent drug targets in themselves?

G protein-coupled receptors (GPCRs) are successfully exploited as drug targets. As our understanding of how distinct GPCR subtypes can be generated expands, so do possibilities for therapeutic intervention via these receptors. Receptor activity-modifying proteins (RAMPs) are excellent examples of proteins that enhance diversity in. GPCR function. They facilitate the creation of binding pockets, controlling the pharmacology of some GPCRs. Moreover, they have the ability to regulate cell-surface trafficking, internalisation and signalling of GPCRs, creating novel opportunities for drug discovery. RAMPs could be directly targeted by drugs, or advantage could be taken of unique RAMP/GPCR interfaces for generating highly selective ligands.

Childhood asthma exacerbations and the Arg16 β2-receptor polymorphism : a meta-analysis stratified by treatment

Peer reviewed

An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.

Toll-like receptor 9 in breast cancer

The innate immune system recognizes microbial features leading to the activation of the adaptive immune system. The role of Toll-like receptor 9 (TLR9) is to recognize microbial DNA. In addition to immune cells, TLR9 is widely expressed in breast cancer in addition to other cancers. Breast cancer is the most common cancer in women, affecting approximately one in eight in industrialized countries. In the clinical setting, breast cancer is divided into three clinical subtypes with type-specific treatments. These subtypes are estrogen receptor (ER)-positive, HER2-positive and triple-negative (TNBC) breast cancer. TNBC is the most aggressive subtype that can be further divided into several subtypes. TNBC tumors lack ER, progesterone receptor and HER2 receptor. Therefore, the current clinically used targeted therapies are not suitable for TNBC treatment as TNBC is a collection of diseases rather than one entity. Some TNBC patients are cured with standard chemotherapy, while others rapidly die due to the disease. There are no clinically used iomarkers which would help in predicting which patients respond to chemotherapy. During this thesis project, we discovered a novel good-prognosis TNBC subtype. These tumors have high TLR9 expression levels. Our findings suggest that TLR9 screening in TNBC patient populations might help to identify the patients that are at the highest risk regarding a relapse. To gain better understanding on the role of TLR9 in TNBC, we developed an animal model which mimicks this disease. We discovered that suppression of TLR9 expression in TNBC cells increases their invasive properties in hypoxia. In line with the clinical findings, TNBC cells with low TLR9 expression also formed more aggressive tumors in vivo. TLR9 expression did not, however, affect TNBC tumor responses to doxorubicin. Our results suggest that tumor TLR9 expression may affect chemotherapyrelated immune responses, however, this requires further investigation. Our other findings revealed that DNA released by chemotherapy-killed cells induces TLR9-mediated invasion in living cancer cells. Normally, extracellular self-DNA is degraded by enzymes, but during massive cell death, for example during chemotherapy, the degradation machinery may be exhausted and self-DNA is taken up into living cells activating TLR9. We also discovered that the malaria drug chloroquine, an inhibitor of autophagy and TLR9 signalling does not inhibit TNBC growth in vivo, independently of the TLR9 status. Finally, we found that ERα as well as the sex hormones estrogen and testosterone regulate TLR9 expression and activity in breast cancer cells in vitro. As a conclusion, we suggest that TLR9 is a potential biomarker in TNBC. ------- Sisäsyntyisen immuniteetin tehtävä on tunnistaa mikrobien molekyylirakenteita, mikä saa aikaan adaptiivisen immuunijärjestelmän aktivoitumisen. Tollin kaltainen reseptori 9 (TLR9) on dna:ta tunnistava sisäsyntyisen immuniteetin reseptori, jota ilmennetään myös useissa syövissä, kuten rintasyövässä. Rintasyöpä on naisten yleisin syöpä, johon joka kahdeksas nainen sairastuu elämänsä aikana. Kliinisesti rintasyöpä jaotellaan kolmeen alatyyppiin, joista kolmoisnegatiivinen rintasyöpä on aggressiivisin. Tämän tyypin syövät eivät ilmennä hormonireseptoreja (estrogeeni- ja progesteronireseptori) tai HER2-reseptoria. Tästä johtuen kolmoisnegatiivisten potilaiden hoitoon ei voida käyttää rintasyövän nykyisten hoitosuositusten mukaisia täsmähoitoja. Kolmoisnegatiivinen rintasyöpä ei kuitenkaan ole yksi sairaus, koska molekyylitasolla sen on osoitettu koostuvan lukuisista, biologialtaan erilaisista syöpämuodoista. Tällä hetkellä kliinisessä käytössä ei ole biomarkkeria, jonka avulla kolmoisnegatiivisen rintasyövän alatyypit voisi erottaa toisistaan. Löysimme uuden kolmoisnegatiivisen syövän alatyypin, joka ilmentää vain vähän TLR9-proteiinia. Tällä alatyypillä on erittäin huono ennuste ja tulostemme perusteella TRL9-tason selvittäminen voisi seuloa huonoennusteiset syövät kolmoisnegatiivisten syöpien joukosta. Kehitimme eläinmallin, jolla voidaan tutkia matalan ja korkean TLR9-tason vaikutuksia kolmoisnegatiivisten kasvainten hoitovasteeseen. Toinen löytömme oli, että kemoterapialla tapettujen syöpäsolujen dna saa aikaan elävien syöpäsolujen TLR9-välitteistä invaasiota. Normaalisti entsyymit hajoittavat yksilön oman solunulkoisen dna:n. Erikoistilanteissa, kuten syöpähoitojen yhteydessä, jolloin solukuolema on massiivista, elimistön oma koneisto ei ehdi tuhoamaan solunulkoista dna:ta ja sitä voi kertyä eläviin soluihin, joissa se aktivoi TLR9:n. Kolmanneksi havaitsimme, että malarialääke klorokiini, joka estää TLR9:n toimintaa ja jolla on syövänvastaisia vaikutuksia soluviljelyolosuhteissa, ei estänyt TLR9-positiivisten tai TLR9-negatiivisten kasvainten kasvua käyttämässämme eläinmallissa. Neljänneksi soluviljelykokeittemme tulokset osoittivat, että sukupuolihormonit estrogeeni ja testosteroni sekä estrogeenireseptori osallistuvat TLR9:n ilmentymisen ja aktiivisuuden säätelyyn. Tuloksemme osoittavat, että TLR9 potentiaalinen biomarkkeri kolmoisnegatiivisessa rintasyövässä.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

International audience

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

Icatibant, An Inhibitor Of Bradykinin Receptor 2, For Hereditary Angioedema Attacks: Prospective Experimental Single-cohort Study.

Hereditary angioedema (HAE) with C1 inhibitor deficiency manifests as recurrent episodes of edema involving the skin, upper respiratory tract and gastrointestinal tract. It can be lethal due to asphyxia. The aim here was to evaluate the response to therapy for these attacks using icatibant, an inhibitor of the bradykinin receptor, which was recently introduced into Brazil. Prospective experimental single-cohort study on the efficacy and safety of icatibant for HAE patients. Patients with a confirmed HAE diagnosis were enrolled according to symptoms and regardless of the time since onset of the attack. Icatibant was administered in accordance with the protocol that has been approved in Brazil. Symptom severity was assessed continuously and adverse events were monitored. 24 attacks in 20 HAE patients were treated (female/male 19:1; 19-55 years; median 29 years of age). The symptoms were: subcutaneous edema (22/24); abdominal pain (15/24) and upper airway obstruction (10/24). The time taken until onset of relief was: 5-10 minutes (5/24; 20.8%); 10-20 (5/24; 20.8%); 20-30 (8/24; 33.4%); 30-60 (5/24; 20.8%); and 2 hours (1/24; 4.3%). The time taken for complete resolution of symptoms ranged from 4.3 to 33.4 hours. Adverse effects were only reported at injection sites. Mild to moderate erythema and/or feelings of burning were reported by 15/24 patients, itching by 3 and no adverse effects in 6. HAE type I patients who received icatibant responded promptly; most achieved improved symptom severity within 30 minutes. Local adverse events occurred in 75% of the patients.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Preserved Fertility In A Patient With Gynecomastia Associated With The P.pro695ser Mutation In The Androgen Receptor.

The androgen insensitivity syndrome (AIS) is described as a dysfunction of the androgen receptor (AR) in 46,XY individuals, which can be associated with mutations in the AR gene or can be due to unknown mechanisms. Different mutations in AIS generally cause variable phenotypes that range from a complete hormone resistance to a mild form usually associated with male infertility. The purpose of this study was to search for mutations in the AR gene in a fertile man with gynecomastia and to evaluate the influence of the mutation on the AR transactivation ability. Sequencing of the AR gene revealed the p.Pro695Ser mutation. It is located within the AR ligand-binding domain. Bioinformatics analysis indicated a deleterious role, which was verified after testing transactivation activity and N-/C-terminal (N/C) interaction by in vitro expression of a reporter gene and 2-hybrid assays. p.Pro695Ser showed low levels of both transactivation activity and N/C interaction at low dihydrotestosterone (DHT) conditions. As the ligand concentration increased, both transactivation activity and N/C interaction also increased and reached normal levels. Therefore, this study provides functional insights for the p.Pro695Ser mutation described here for the first time in a patient with mild AIS. The expression profile of p.Pro695Ser not only correlates to the patient's phenotype, but also suggests that a high-dose DHT therapy may overcome the functional deficit of the mutant AR.

[the New Classification Of Breast Cancers: Finding The Luminal A].

To compare the distributions of patients with clinical-pathological subtypes of luminal B-like breast cancer according to the 2011 and 2013 St. Gallen International Breast Cancer Conference Expert Panel. We studied 142 women with breast cancer who were positive to estrogen receptor and had been treated in São Paulo state, southeast Brazil. The expression of the following receptors was assessed by immunohistochemistry: estrogen, progesterone (PR) and Ki-67. The expression of HER-2 was measured by fluorescent in situ hybridization analysis in tissue microarray. There were 29 cases of luminal A breast cancers according to the 2011 St. Gallen International Breast Cancer Conference Expert Panel that were classified as luminal B-like in the 2013 version. Among the 65 luminal B-like breast cancer cases, 29 (45%) were previous luminal A tumors, 15 cases (20%) had a Ki-67 >14% and were at least 20% PR positive and 21 cases (35%) had Ki-67 >14% and more than 20% were PR positive. The 2013 St. Gallen consensus updated the definition of intrinsic molecular subtypes and increased the number of patients classified as having luminal B-like breast cancer in our series, for whom the use of cytotoxic drugs will probably be proposed with additional treatment cost.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.