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1.
Mar Drugs ; 17(10)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627357

RESUMO

Increasingly cancer is being viewed as a channelopathy because the passage of ions via ion channels and transporters mediate the regulation of tumor cell survival, death, and motility. As a result, a potential targeted therapy for cancer is to use venom peptides that are selective for ion channels and transporters overexpressed in tumor cells. Here we describe the selectivity and mechanism of action of terebrid snail venom peptide, Tv1, for treating the most common type of liver cancer, hepatocellular carcinoma (HCC). Tv1 inhibited the proliferation of murine HCC cells and significantly reduced tumor size in Tv1-treated syngeneic tumor-bearing mice. Tv1's mechanism of action involves binding to overexpressed transient receptor potential (TRP) channels leading to calcium dependent apoptosis resulting from down-regulation of cyclooxygenase-2 (COX-2). Our findings demonstrate the importance of modulating ion channels and the unique potential of venom peptides as tumor specific ligands in the quest for targeted cancer therapies.


Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Venenos de Moluscos/farmacologia , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
2.
Diving Hyperb Med ; 49(3): 225-228, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31523798

RESUMO

INTRODUCTION: Between 40,000 and 50,000 divers and swimmers are envenomated each year and diving as a hobby is becoming increasingly popular. In the Mediterranean, envenomation is most often by Weever fish, Scorpion fish and jellyfish but coral and sea urchins may also be venomous. ENVENOMATION: Most stings cause local inflammation, oedema and pain. The severity of pain varies with the venom and the amount injected. In severe cases, stings may be life-threatening due to cardiogenic or anaphylactic shock or penetration of vital structures. MANAGEMENT: Most cases of envenomation are preventable with a combination of measures including the avoidance of contact through good buoyancy control, the wearing of body-suits, and by maintaining visual awareness. Immediate management is to return to the surface, elevate and wash the site of injury. Immersion in hot water followed by simple analgesics for pain relief has been shown to be more effective than other methods. More severe cases should be identified by symptoms including confusion and heavy bleeding and referred to qualified medical care. CONCLUSION: Envenomation by subaquatic species is common and preventable yet the dissemination of the appropriate knowledge is limited. This knowledge summary provides pertinent information aimed at divers in preventing and managing such injuries.


Assuntos
Antivenenos/uso terapêutico , Mordeduras e Picadas , Mergulho , Primeiros Socorros/métodos , Animais , Mordeduras e Picadas/complicações , Mordeduras e Picadas/terapia , Serviços Médicos de Emergência , Venenos de Peixe/envenenamento , Humanos , Toxinas Marinhas/envenenamento , Venenos de Moluscos/envenenamento , Cifozoários
3.
Proc Natl Acad Sci U S A ; 116(37): 18700-18709, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31444298

RESUMO

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.


Assuntos
Membrana Celular/efeitos dos fármacos , Proteínas de Drosophila/antagonistas & inibidores , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio Kv1.2/antagonistas & inibidores , Venenos de Moluscos/toxicidade , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Desenho de Fármacos , Feminino , Ligação de Hidrogênio/efeitos dos fármacos , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/isolamento & purificação , Canal de Potássio Kv1.2/metabolismo , Dose Letal Mediana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Venenos de Moluscos/química , Mutação , Oócitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/isolamento & purificação , Superfamília Shaker de Canais de Potássio/metabolismo , Água/química , Água/metabolismo , Xenopus laevis
4.
Mol Biol Rep ; 46(5): 5479-5486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31102148

RESUMO

Constant research into the pharmaceutical properties of marine natural products has led to the discovery of many potentially active agents considered worthy of medical applications. Genus Conus, which approximately comprises 700 species, is currently under every researcher's interest because of the conopeptides in their crude venom. Conopeptides have a wide range of pharmacological classes and properties. This research focused on the crude venom of Conus striatus to assess its analgesic activity, mutagenicity, nephrotoxicity, and hepatotoxicity in mice. The crude venom was extracted from the conus snails and the protein concentration was determined using Bradford's method. The analgesic activity of the venom was determined using the hot-plate method and standard IFCC method was used to determine the alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Evaluation of mutagenicity was done using micronucleus assay and the nephrotoxicity of the venom was determined using Kidney Coefficient and serum creatinine concentration. The maximum tolerable dose (MTD) of the crude venom was found to be 75 ppm. The venom exhibited potent analgesic activity even higher than the positive control (Ibuprofen). Most of the analgesic drugs can usually impact damage in the liver and kidneys. However, AST and ALT results revealed that the venom has no adverse effects on the liver. Although the venom increased the incidence of micronucleated polychromatic erythrocytes, making it mutagenic, with MTD concentration's mutagenicity comparable to the positive control methyl methanesulfonate (MMS). The kidney coefficients, on the other hand, showed no significant difference between the treated groups and that of the untreated group. The serum creatinine also showed a concentration-dependent increase; with MTD treated mice got the highest creatinine concentration. However, MTD/2 and MTD/4 showed no significant difference in creatinine levels with respect to the untreated groups. Hence, the nephrotoxicity of the venom was only evident when used at higher concentration. The venom exhibited potent analgesic activity indicated that the C. striatus crude venom extract could have a potential therapeutic component as analgesic drugs that displayed no hepatic damage. This study also suggests that for this venom to be utilized for future medical applications, their usage must be regulated and properly monitored to avoid nephrotoxic effect.


Assuntos
Venenos de Moluscos/metabolismo , Venenos de Moluscos/farmacologia , Alanina Transaminase/sangue , Analgésicos/metabolismo , Analgésicos/farmacologia , Animais , Aspartato Aminotransferases/sangue , Caramujo Conus , Creatinina/sangue , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peçonhas/metabolismo
5.
J Biol Chem ; 294(22): 8745-8759, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30975904

RESUMO

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.


Assuntos
Conotoxinas/química , Caramujo Conus/metabolismo , Cisteína/química , Granulinas/química , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Conotoxinas/metabolismo , Dissulfetos/química , Granulinas/metabolismo , Espectroscopia de Ressonância Magnética , Venenos de Moluscos/metabolismo , Conformação Proteica em Folha beta , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
6.
Biochem Pharmacol ; 164: 342-348, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028742

RESUMO

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.


Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/fisiologia , Venenos de Moluscos/farmacologia , Neuropeptídeos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/fisiologia , Bloqueadores do Canal Iônico Sensível a Ácido/isolamento & purificação , Animais , Caramujo Conus , Relação Dose-Resposta a Droga , Feminino , Humanos , Venenos de Moluscos/isolamento & purificação , Neuropeptídeos/isolamento & purificação , Antagonistas Nicotínicos/isolamento & purificação , Xenopus laevis
7.
Mar Drugs ; 17(3)2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875751

RESUMO

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.


Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Caramujo Conus/química , Fluorenos/química , Humanos , Venenos de Moluscos/química , Peptídeos/síntese química
8.
Mol Cell Proteomics ; 18(5): 876-891, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30765458

RESUMO

The phospholipase A2 (PLA2s) superfamily are ubiquitous small enzymes that catalyze the hydrolysis of phospholipids at the sn-2 ester bond. PLA2s in the venom of cone snails (conodipines, Cdpi) are composed of two chains termed as alpha and beta subunits. Conodipines are categorized within the group IX of PLA2s. Here we describe the purification and biochemical characterization of three conodipines (Cdpi-P1, -P2 and -P3) isolated from the injected venom of Conus purpurascens Using proteomics methods, we determined the full sequences of all three conodipines. Conodipine-P1-3 have conserved consensus catalytic domain residues, including the Asp/His dyad. Additionally, these enzymes are expressed as a mixture of proline hydroxylated isoforms. The activities of the native Conodipine-Ps were evaluated by conventional colorimetric and by MS-based methods, which provide the first detailed cone snail venom conodipine activity monitored by mass spectrometry. Conodipines can have medicinal applications such inhibition of cancer proliferation, bacterial and viral infections among others.


Assuntos
Caramujo Conus/química , Venenos de Moluscos/química , Fosfolipases A2/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Fracionamento Químico , Galinhas , Gema de Ovo/metabolismo , Humanos , Injeções , Peso Molecular , Proteólise , Proteômica , Solubilidade , Transcriptoma/genética
9.
Elife ; 82019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30747102

RESUMO

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.


Assuntos
Caramujo Conus/química , Insulina/metabolismo , Venenos de Moluscos/metabolismo , Venenos/metabolismo , Receptor de Insulina/agonistas , Animais , Antígenos CD/química , Modelos Animais de Doenças , Humanos , Hipoglicemia/patologia , Insulina/química , Insulina/genética , Camundongos , Simulação de Dinâmica Molecular , Envenenamento/patologia , Receptor de Insulina/química , Peixe-Zebra
10.
Elife ; 82019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30747103

RESUMO

Cone snails have evolved a variety of insulin-like molecules that may help with the development of better treatments for diabetes.


Assuntos
Diabetes Mellitus , Peçonhas , Animais , Ligantes , Venenos de Moluscos , Peptídeos , Receptor de Insulina , Vertebrados
11.
J Proteomics ; 190: 12-20, 2019 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-29777871

RESUMO

There have been numerous attempts to develop non-opioid drugs for severe pain, but the vast majority of these efforts have failed. A notable exception is Ziconotide (Prialt®), approved by the FDA in 2004. In this review, we summarize the present status of Ziconotide as a therapeutic drug and introduce a wider framework: the potential of venom peptides from cone snails as a resource providing a continuous pipeline for the discovery of non-opioid pain therapeutics. An auxiliary theme that we hope to develop is that these venoms, already a validated starting point for non-opioid drug leads, should also provide an opportunity for identifying novel molecular targets for future pain drugs. This review comprises several sections: the first focuses on Ziconotide as a therapeutic (including a historical retrospective and a clinical perspective); followed by sections on other promising Conus venom peptides that are either in clinical or pre-clinical development. We conclude with a discussion on why the outlook for discovery appears exceptionally promising. The combination of new technologies in diverse fields, including the development of novel high-content assays and revolutionary advancements in transcriptomics and proteomics, puts us at the cusp of providing a continuous pipeline of non-opioid drug innovations for pain. SIGNIFICANCE: The current opioid epidemic is the deadliest drug crisis in American history. Thus, this review on the discovery of non-opioid pain therapeutics and pathways from cone snail venoms is significant and timely.


Assuntos
Analgésicos não Entorpecentes/uso terapêutico , Descoberta de Drogas , Venenos de Moluscos/uso terapêutico , Dor/tratamento farmacológico , ômega-Conotoxinas/uso terapêutico , Animais , Caramujo Conus/patogenicidade , Venenos de Moluscos/química , Dor/prevenção & controle , Manejo da Dor/métodos , Proteômica
12.
Int J Biol Macromol ; 124: 1145-1155, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30521901

RESUMO

A low molecular weight posterior salivary gland (PSG) toxin was isolated and purified from the cuttlefish Sepia prashadi by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The protein and neutral sugar content of the PSG toxin was determined to be 1.033 mg/g and 282 µg/g. Fourier Transform Infrared (FT-IR) spectroscopy revealed the presence of υ-OH, υ-CO and δ-NH functional groups. Circular Dichroism (CD) spectroscopy and K2D2 analysis quantified the presence of 38.39% α-helix and 9.25% ß-sheet and 52.36% of ß-turn. Matrix Assisted Laser Desorption/Ionization-Time-of Flight/Mass Spectrometry (MALDI-TOF/MS) and MASCOT analysis revealed the amino acid sequence of MEMQSKQQNSKAPANRKIFPWMKTSAVATASKRVEMASLLNLQERQIKIWFQNRMKQKSQQPQTR (1.92 kDa) homologous to homeobox protein H4 of pufferfish, T. rubripes. The PSG toxin showed differential stability with pH and induced premature hatching in Zebrafish eggs and dose dependant developmental malformations in embryos with a Maximum tolerated dose of 1.85 µM. The PSG toxin exhibited significant antibacterial activity with pronounced zone of inhibition against S. typhimurium (12.94 mm) and inhibited avian RBC binding of Newcastle Disease virus (NDV) at a titre value of 1/4. The present study strongly advocates the biomedical potential of the PSG toxin from S. prashadi and illustrates its promise as a potential avian antimicrobial agent of the future.


Assuntos
Antibacterianos/farmacologia , Antivirais/farmacologia , Decapodiformes/química , Glicoproteínas/farmacologia , Venenos de Moluscos/farmacologia , Glândulas Salivares/química , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antivirais/química , Antivirais/isolamento & purificação , Galinhas , Embrião não Mamífero/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/virologia , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Peso Molecular , Venenos de Moluscos/química , Venenos de Moluscos/isolamento & purificação , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/fisiologia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Estabilidade Proteica , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ligação Viral/efeitos dos fármacos , Peixe-Zebra , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento
13.
Sci Rep ; 8(1): 15276, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323294

RESUMO

The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, we developed a sequence driven:activity-based hybrid approach for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, we characterized the physiological and behavioral phenotypes of two peptides from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. Our results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 µM) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 µM) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet.


Assuntos
Comportamento Animal/efeitos dos fármacos , Drosophila/efeitos dos fármacos , Venenos de Moluscos/química , Peptídeos/química , Animais , Comportamento Animal/fisiologia , Drosophila/fisiologia , Venenos de Moluscos/farmacologia , Venenos de Moluscos/toxicidade , Peptídeos/farmacologia , Peptídeos/toxicidade , Caramujos/química , Caramujos/genética , Transcriptoma/genética
14.
Protein Pept Lett ; 25(11): 1035-1040, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30345912

RESUMO

BACKGROUND: Conotoxins are neuro-pharmacologically active cysteine rich peptides isolated from the venom complex of marine cone snails. These are usually made of even number of cysteines. METHOD: In this study we characterised six novel conotoxin sequences from the venom of Conus leopardus collected from the Andaman Sea, namely Le907 (C-C), Le868 (C-C), Le933 (-C-CC), Le949 (-C-CC), Le1988 (C-C-CC-C) and Le1642 (CC-C-C) using de novo mass spectrometrybased sequencing methods. Astonishingly 3 of these peptides possess novel arrangements of cysteine residues with odd number of cysteines (-C-CC; C-C-CC-C), namely Le933, Le949 and Le1988. Further, a post-translational variant of peptide Le933 was identified and experimentally determined to contain hydroxyproline. RESULTS: The unusual cysteine arrangements observed suggests novel class of conotoxins. These results expand our understanding of the diversity of odd cysteine arrangements in conotoxins.


Assuntos
Conotoxinas/química , Caramujo Conus , Cisteína , Venenos de Moluscos/química , Sequência de Aminoácidos , Animais
15.
Toxicon ; 154: 28-34, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30243794

RESUMO

Members of Mas related G-protein coupled receptors (Mrgpr) are known to mediate itch. To date, several compounds have been shown to activate these receptors, including chloroquine, a common antimalarial drug, and peptides of the RF-amide family. However, specific ligands for these receptors are still lacking and there is a need for novel compounds that can be used to modulate the receptors in order to understand the cellular and molecular mechanism in which they mediate itch. Some cone snail venoms were previously shown to induce itch in mice. Here, we show that the venom of Conus textile induces itch through activation of itch-sensing sensory neurons, marked by their sensitivity to chloroquine. Two RF-amide peptides, CNF-Tx1 and CNF-Tx2, were identified in a C. textile venom gland transcriptome. These belong to the conorfamide family of peptides which includes previously described peptides from the venoms of Conus victoriae (CNF-Vc1) and Conus spurius (CNF-Sr1 and CNF-Sr2). We show that CNF-Vc1 and CNF-Sr1 activate MrgprC11 whereas CNF-Vc1 and CNF-Tx2 activate the human MrgprX1 (hMrgprX1). The peptides CNF-Tx1 and CNF-Sr2 do not activate MrgprC11 or hMrgprX1. Intradermal injection of CNF-Vc1 and CNF-Tx2 into the cheek of a transgenic mouse expressing hMrgprX1 instead of endogenous mouse Mrgprs resulted in itch-related scratching thus demonstrating the in vivo activity of these peptides. Using truncated analogues of CNF-Vc1, we identified amino acids at positions 7-14 as important for activity against hMrgprX1. The conopeptides reported here are tools that can be used to advance our understanding of the cellular and molecular mechanism of itch mediated by Mrgprs.


Assuntos
Conotoxinas/farmacologia , Prurido/induzido quimicamente , Prurido/fisiopatologia , Receptores Acoplados a Proteínas-G/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Masculino , Camundongos , Venenos de Moluscos/farmacologia , Neuropeptídeos/farmacologia , Peptídeos , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/biossíntese , Receptores Acoplados a Proteínas-G/genética , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Transcriptoma
16.
J Proteome Res ; 17(11): 3866-3876, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30220204

RESUMO

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Venenos de Moluscos/genética , Octopodiformes/genética , Proteogenômica/métodos , Saliva/metabolismo , Transcriptoma , Animais , Feminino , Ontologia Genética , Larva/química , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Anotação de Sequência Molecular , Venenos de Moluscos/classificação , Venenos de Moluscos/metabolismo , Neurotoxinas/classificação , Neurotoxinas/genética , Neurotoxinas/metabolismo , Octopodiformes/química , Octopodiformes/crescimento & desenvolvimento , Octopodiformes/metabolismo , Filogenia , Proteoma/genética , Proteoma/metabolismo , Saliva/química , Glândulas Salivares/química , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Serina Proteases/classificação , Serina Proteases/genética , Serina Proteases/metabolismo
17.
Toxicon ; 151: 129-136, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30026171

RESUMO

In recent years, the neurotoxin ß-N-methylamino-L-alanine (BMAA) has been reported in some marine mollusk species. To further discover BMAA in marine animals, a total of 59 samples belonging to 3 phyla, 22 families, and 43 species, were collected from Dalian, Rongcheng, and Zhoushan cities, China, in April 2017. All samples were quantified by a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) analysis of underivatized extract, and ten samples were also analyzed by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using a precolumn AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate)-derivatization method. Results indicated that 48 mollusk samples contained BMAA with concentrations above the limit of detection (0.31 µg g-1 wet weight), and the isomers of BMAA, ß-amino-N-methylalanine (BAMA) and 2,4-diaminobutyric acid (DAB) were universally present in most samples. However, N-(2-aminoethyl) glycine (AEG) was not found in any sample. Comparison of both analytical methods showed that BMAA and BAMA were not completely separated by the HILIC column although they still could be identified by specific transitions. In contrast the C18 column provided good separation for the AQC-derivatives of BMAA and all of its isomers. Development of analytical methods and stable isotope tracing of BMAA should be carried out in the future.


Assuntos
Diamino Aminoácidos/metabolismo , Espectrometria de Massas , Moluscos/fisiologia , Venenos de Moluscos/química , Diamino Aminoácidos/química , Animais , Estrutura Molecular
18.
Mol Pharmacol ; 94(4): 1114-1124, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30012583

RESUMO

Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na+ channel with important roles in pain. ASIC3 quickly desensitizes in less than a second, limiting its capacity to sense sustained acidosis during pain. RFamide neuropeptides are modulators of ASIC3 that slow its desensitization and induce a variable sustained current. The molecular mechanism of slowed desensitization and the RFamide binding site on ASIC3 are unknown. RPRFamide, a RFamide from the venom of a cone snail, has a comparatively high affinity for ASIC3 and strongly slows its desensitization. Here we show that covalent binding of a UV-sensitive RPRFamide variant to ASIC3 prevents desensitization, suggesting that RPRFamide has to unbind from ASIC3 before it can desensitize. Moreover, we show by in silico docking to a homology model of ASIC3 that a cavity in the lower palm domain, which is also known as the nonproton ligand-sensing domain, is a potential binding site of RPRFamide. Finally, using extensive mutagenesis of residues lining the nonproton ligand-sensing domain, we confirm that this domain is essential for RPRFamide modulation of ASIC3. As comparative analysis of ASIC crystal structures in the open and in the desensitized conformation suggests that the lower palm domain contracts during desensitization, our results collectively suggest that RPRFamide, and probably also other RFamide neuropeptides, bind to the nonproton ligand-sensing domain to stabilize the open conformation of ASIC3.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Venenos de Moluscos/farmacologia , Neuropeptídeos/farmacologia , Animais , Feminino , Ligantes , Dor/tratamento farmacológico , Conformação Proteica , Domínios Proteicos , Prótons , Ratos , Xenopus laevis/metabolismo
19.
Mar Drugs ; 16(6)2018 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899286

RESUMO

Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus/metabolismo , Venenos de Moluscos/química , Neurônios/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Conotoxinas/química , Sistemas de Liberação de Medicamentos/métodos , Conformação Molecular , Neurônios/metabolismo , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Relação Estrutura-Atividade
20.
Sci Rep ; 8(1): 330, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29321522

RESUMO

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.


Assuntos
Caramujo Conus/fisiologia , Venenos de Moluscos/metabolismo , Peptídeos/metabolismo , Comportamento Predatório , Proteômica , Animais , Cromatografia Líquida , Conotoxinas/genética , Conotoxinas/metabolismo , Expressão Gênica , Venenos de Moluscos/genética , Peptídeos/genética , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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