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1.
Mol Immunol ; 119: 144-153, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32023500

RESUMO

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.


Assuntos
Anticorpos Neutralizantes/imunologia , Crotoxina/imunologia , Neurotoxinas/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Antivenenos/genética , Antivenenos/imunologia , Crotoxina/química , Crotoxina/genética , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Feminino , Camundongos , Modelos Moleculares , Neurotoxinas/química , Neurotoxinas/genética , Engenharia de Proteínas , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
2.
Sci Total Environ ; 703: 135513, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31761374

RESUMO

ß-N-Methylamino-l-alanine (BMAA), a new cyanobacterial toxin, is found in different aquatic ecosystems worldwide and is to threaten the human nervous system. Therefore, it is important for water plants to develop feasible methods to counter the effects of BMAA. In this study, the removal of BMAA by chlorine, as well as its intermediate products, at different pH values and the mechanism of pH on the removal BMAA were investigated. The results showed that the chlorination of BMAA is in accordance with the second-order kinetics model. The reaction rate of chlorinated BMAA increased with the increase in the concentration of chlorine. The pH of the solution significantly affected the reaction rate. The apparent kinetic constant (kapp) decreased from 6.00 × 103 M-1·min-1 to 35.5 M-1·min-1 when the pH increased from 4.5 to 9 in the chlorine concentration of 32.23 µM. It is probable that the species distribution and proportion of BMAA and chlorine at different pH values were the main causes of this phenomenon. Additionally, the chlorination reaction consisted of four elementary reactions and hydrogen ions were beneficial to the reaction. The temperature also affected the reaction rate and the activation energy of the reaction was 16.6 ± 1.99 kJ·M-1. A variety of degradation products were detected and the path of degradation was speculated. Chlorination, dechlorination, and decarboxylation were the main processes of oxidative degradation. Furthermore, the composition of the degradation products was the same at different pH values.


Assuntos
Diamino Aminoácidos/química , Cloro/química , Poluentes Químicos da Água/química , Purificação da Água/métodos , Diamino Aminoácidos/análise , Halogenação , Neurotoxinas/química , Poluentes Químicos da Água/análise
3.
Dokl Biochem Biophys ; 487(1): 251-255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31559591

RESUMO

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Neurotoxinas/química , Antagonistas Nicotínicos/química , Polissacarídeos/química , Receptores Nicotínicos/metabolismo , Sulfatos/química , Cápsulas , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Tamanho da Partícula , Venenos de Serpentes/química
4.
Nat Commun ; 10(1): 3642, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409779

RESUMO

Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus, Dendroaspis, Naja, Walterinnesia, Ophiophagus and Hydrophis. These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.


Assuntos
Anticorpos/imunologia , Venenos Elapídicos/imunologia , Elapidae/imunologia , Neurotoxinas/imunologia , Sequência de Aminoácidos , Animais , Venenos Elapídicos/genética , Elapidae/genética , Cavalos , Imunização , Masculino , Camundongos , Neurotoxinas/química , Neurotoxinas/genética , Alinhamento de Sequência
5.
J Environ Sci Health B ; 54(11): 883-891, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31311415

RESUMO

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.


Assuntos
Anelídeos/enzimologia , Inibidores da Colinesterase/toxicidade , Colinesterases/química , Neurotoxinas/toxicidade , Animais , Anelídeos/efeitos dos fármacos , Biomarcadores/química , Carbamatos/química , Carbaril/química , Carbaril/toxicidade , Carbofurano/química , Carbofurano/toxicidade , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Inibidores da Colinesterase/química , Colinesterases/metabolismo , Cinética , Metomil/química , Metomil/toxicidade , Neurotoxinas/química
6.
Toxicon ; 168: 147-157, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31330191

RESUMO

Voltage gated ion channels have become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to anti-inflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel toxins. A putative novel sodium channel agonist (molecular mass of 4619.7 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii bio-assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel agonist/inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated the presence of multiple disulfide bonds in a tight ß-defensin cystine conformation. An IC50 value of 26 nM was determined for total channel inhibition on MCF-7 cells. The unique putative sodium channel agonist initiating with a cystine bond indicates a divergent evolution to those previously isolated from Bunodosoma species.


Assuntos
Anêmonas-do-Mar/química , Agonistas de Canais de Sódio/química , Sequência de Aminoácidos , Animais , Artemia , Humanos , Células MCF-7 , Toxinas Marinhas , Neurotoxinas/química , Peptídeos/química , África do Sul
7.
Int J Biol Macromol ; 134: 613-621, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31071401

RESUMO

Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25-200 µg/mL) - the main component of Crotalus durissus terrificus snake venom - interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 µg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1ß), as well as protected cells against H2O2-induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies.


Assuntos
Crotoxina/química , Crotoxina/farmacologia , Células Endoteliais/efeitos dos fármacos , Neurotoxinas/química , Neurotoxinas/farmacologia , Venenos de Serpentes/química , Venenos de Serpentes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Citocinas/biossíntese , Humanos , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos
8.
Int J Pharm ; 563: 91-100, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30890451

RESUMO

This study is directed towards the gentle transdermal delivery of Neurotoxin (NT) and study of the treatment of Rheumatoid Arthritis (RA) in rats by NT loaded dissolving Microneedles (DMNs-NT). The DMNs-NT fabrication involved a two-step centrifugation method. The quadrangular pyramid shape needles had great mechanical strength. The upper part of the needle contained 15.4 ±â€¯0.5 µg of drug per patch. Blank DMNs showed favorable biocompatibility and low toxicity on the chondrocyte cells. Both NT and DMNs-NT displayed anti-inflammatory capabilities ex-vitro. The results of ex-vitro evaluation of DMNs the skin penetration depth of DMNs-NT rats was higher than 70 µm and the cumulative penetration of NT in DMNs could reach 95.8% in 4 h, whereas, the NT solution could barely penetrate the skin, thereby proving the favorable facilitation of NT transdermal delivery. The needle structure dissolved completely after 10 min in vivo and the channel on the Stratum Corneum (SC) was closed after 6 h. There was no significant adverse reaction on the skin after 15 days of administration. The pharmacodynamic study showed that DMNs-NT significantly reduced the toe swelling of RA rats and reduced the levels of TNF-α and IL-1ß in serum to alleviate the injury of the ankle joints. DMNs-NT held favorable stability in 3 months. All these results established that DMNs-NT could penetrate the skin of rats in a biocompatible manner, and have a strong therapeutic effect on rat RA by transdermal delivery.


Assuntos
Anti-Inflamatórios/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Microinjeções , Agulhas , Neurotoxinas/administração & dosagem , Administração Cutânea , Animais , Anti-Inflamatórios/química , Artrite Reumatoide/sangue , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , Interleucina-1beta/sangue , Neurotoxinas/química , Ratos Wistar , Pele/metabolismo , Absorção Cutânea , Solubilidade , Fator de Necrose Tumoral alfa/sangue
9.
Toxins (Basel) ; 11(3)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857180

RESUMO

Cobra venom cardiotoxins (CVCs) can translocate to mitochondria to promote apoptosis by eliciting mitochondrial dysfunction. However, the molecular mechanism(s) by which CVCs are selectively targeted to the mitochondrion to disrupt mitochondrial function remains to be elucidated. By studying cardiotoxin from Naja mossambica mossambica cobra (cardiotoxin VII4), a basic three-fingered S-type cardiotoxin, we hypothesized that cardiotoxin VII4 binds to cardiolipin (CL) in mitochondria to alter mitochondrial structure/function and promote neurotoxicity. By performing confocal analysis, we observed that red-fluorescently tagged cardiotoxin rapidly translocates to mitochondria in mouse primary cortical neurons and in human SH-SY5Y neuroblastoma cells to promote aberrant mitochondrial fragmentation, a decline in oxidative phosphorylation, and decreased energy production. In addition, by employing electron paramagnetic resonance (EPR) and protein nuclear magnetic resonance (¹H-NMR) spectroscopy and phosphorescence quenching of erythrosine in model membranes, our compiled biophysical data show that cardiotoxin VII4 binds to anionic CL, but not to zwitterionic phosphatidylcholine (PC), to increase the permeability and formation of non-bilayer structures in CL-enriched membranes that biochemically mimic the outer and inner mitochondrial membranes. Finally, molecular dynamics simulations and in silico docking studies identified CL binding sites in cardiotoxin VII4 and revealed a molecular mechanism by which cardiotoxin VII4 interacts with CL and PC to bind and penetrate mitochondrial membranes.


Assuntos
Proteínas Cardiotóxicas de Elapídeos/toxicidade , Membranas Mitocondriais/efeitos dos fármacos , Neurotoxinas/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Cardiotóxicas de Elapídeos/química , Feminino , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Naja , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/química , Gravidez , Transporte Proteico
10.
Toxins (Basel) ; 11(2)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717279

RESUMO

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum. This enzyme, which is Ca2+-dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA2´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA2 enzymes.


Assuntos
Antozoários , Córtex Motor/efeitos dos fármacos , Síndromes Neurotóxicas , Neurotoxinas/toxicidade , Fosfolipases A2/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Atividade Motora/efeitos dos fármacos , Córtex Motor/patologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Ratos Wistar
11.
Chembiochem ; 20(10): 1231-1241, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30605564

RESUMO

Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage-gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.


Assuntos
Neurotoxinas/biossíntese , Bloqueadores dos Canais de Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Ligantes , Estrutura Molecular , Neurotoxinas/química , Plantas/química , Bloqueadores dos Canais de Sódio/química
12.
Ecotoxicol Environ Saf ; 172: 72-81, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30682636

RESUMO

Environmental exposure to the amino acid ß-methylamino-L-alanine (BMAA) was linked to the high incidence of neurodegenerative disease first reported on the island of Guam in the 1940s and has more recently been implicated in an increased incidence of amyotrophic lateral sclerosis (ALS) in parts of the USA. BMAA has been shown to be produced by a range of cyanobacteria and some marine diatoms and dinoflagellates in different parts of the world. BMAA is commonly found with two of its constitutional isomers: 2,4- diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl) glycine (AEG). These isomers are thought to be co-produced by the same organisms that produce BMAA and MS/MS analysis following LC separation can add an additional level of specificity over LC-FL. Although the presence of BMAA and 2,4-DAB in surface scum samples from several sites in Australia has been reported, which Australian cyanobacterial species are capable of BMAA, 2,4-DAB and AEG production remains unknown. The aims of the present studies were to identify some of the cyanobacterial genera or species that can produce BMAA, 2,4-DAB and AEG in freshwater cyanobacteria blooms in eastern Australia. Eleven freshwater sites were sampled and from these, 19 single-species cyanobacterial cultures were established. Amino acids were extracted from cyanobacterial cultures and analysed using liquid chromatography-tandem mass spectrometry. BMAA was detected in 17 of the 19 isolates, 2,4-DAB was detected in all isolates, and AEG was detected in 18 of the 19 isolates, showing the prevalence of these amino acids in Australian freshwater cyanobacteria. Concentrations of all three isomers in Australian cyanobacteria were generally higher than the concentrations reported elsewhere. This study confirmed the presence of BMAA and its isomers in cyanobacteria isolated from eastern Australian freshwater systems, and determined which Australian cyanobacterial genera or species were capable of producing them when cultured under laboratory conditions.


Assuntos
Diamino Aminoácidos/análise , Diamino Aminoácidos/química , Cianobactérias/química , Aminoácidos/análise , Austrália , Cromatografia Líquida , Água Doce/microbiologia , Glicina/análise , Glicina/química , Isomerismo , Neurotoxinas/análise , Neurotoxinas/química , Espectrometria de Massas em Tandem
13.
J Proteomics ; 192: 196-207, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30205237

RESUMO

Venoms of the three species of Ophryacus (O. sphenophrys, O. smaragdinus, and O. undulatus), a viperid genus endemic to Mexico, were analyzed for the first time in the present work. The three venoms lacked procoagulant activity on human plasma, but induced hemorrhage and were highly lethal to mice. These venoms also displayed proteolytic and phospholipase A2 activities in vitro. The venom of O. sphenophrys was the most lethal and caused hind-limb paralysis in mice. Proteomic profiling of O. sphenophrys venom showed a predominance of metalloproteinase (34.9%), phospholipase A2 (24.8%) and serine protease (17.1%) in its composition. Strikingly, within its PLA2 components, 12.9% corresponded to a Crotoxin-like heterodimer, here named Sphenotoxin, which was not found in the other two species of Ophryacus. Sphenotoxin, like Crotoxin, is composed of non-covalently bound A and B subunits. Partial amino acid sequence was obtained for Sphenotoxin B and was similar (78-89%) to other subunits described. The mouse i.v. LD50 of Sphenotoxin at 1:1 M radio was 0.16 µg/g. Also, like Crotoxin, Sphenotoxin induced a potent neuromuscular blockade in the phrenic nerve-diaphragm preparation. Ophryacus is the fifth genus and O. sphenophrys the third non-rattlesnake species shown to contain a novel Crotoxin-like heterodimeric ß-neurotoxin. BIOLOGICAL SIGNIFICANCE: Ophryacus is an endemic genus of semi-arboreal pitvipers from Mexico that includes three species with restricted distributions. Little is known about the natural history of these species and nothing is known about the properties of their venoms. Research on these species' venoms could generate relevant information regarding venom composition of Mexican pitvipers. Additionally, research into the presence of neurotoxic Crotoxin-like molecules outside of rattlesnakes (genera Crotalus and Sistrurus) has identified this molecule in several new genera. Knowing which genera and species possess neurotoxic components is important to fully understand the repercussions of snakebites, the interaction with prey and predators, and the origin, evolution, and phylogenetic distribution of Crotoxin-like molecules during the evolutionary history of pitvipers. Our study expands current knowledge regarding venom's compositions and function from Mexican pitvipers, providing a comparative venom characterization of major activities in the three Ophryacus species. Additionally, the discovery and characterization of a novel Crotoxin-like molecule, here named Sphenotoxin, in O. sphenophrys, and the detailed protein composition of O. sphenophrys venom supports the hypotheses that Crotoxin-like -ß-neurotoxins are more widespread than initially thought.


Assuntos
Crotalinae/metabolismo , Crotoxina , Neurotoxinas , Multimerização Proteica , Animais , Crotalinae/classificação , Crotoxina/química , Crotoxina/metabolismo , Crotoxina/toxicidade , Humanos , México , Camundongos , Neurotoxinas/química , Neurotoxinas/toxicidade , Especificidade da Espécie
14.
Nat Commun ; 9(1): 5367, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560862

RESUMO

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Membranas Intracelulares/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Sequência de Aminoácidos , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/isolamento & purificação , Cristalografia por Raios X , Citosol/metabolismo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Modelos Moleculares , Neurônios/citologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/química
15.
Proc Natl Acad Sci U S A ; 115(50): E11837-E11846, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30463948

RESUMO

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel's outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation.


Assuntos
Capsaicina/toxicidade , Neurotoxinas/toxicidade , Canais de Cátion TRPV/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurotoxinas/química , Neurotoxinas/genética , Técnicas de Patch-Clamp , Venenos de Aranha/toxicidade , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética
16.
Mar Drugs ; 16(11)2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30380764

RESUMO

Cone snail venoms provide an ideal resource for neuropharmacological tools and drug candidates discovery, which have become a research hotspot in neuroscience and new drug development. More than 1,000,000 natural peptides are produced by cone snails, but less than 0.1% of the estimated conotoxins has been characterized to date. Hence, the discovery of novel conotoxins from the huge conotoxin resources with high-throughput and sensitive methods becomes a crucial key for the conotoxin-based drug development. In this review, we introduce the discovery methodology of new conotoxins from various Conus species. It focuses on obtaining full N- to C-terminal sequences, regardless of disulfide bond connectivity through crude venom purification, conotoxin precusor gene cloning, venom duct transcriptomics, venom proteomics and multi-omic methods. The protocols, advantages, disadvantages, and developments of different approaches during the last decade are summarized and the promising prospects are discussed as well.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Neurotoxinas/farmacologia , Animais , Clonagem Molecular/métodos , Conotoxinas/química , Conotoxinas/isolamento & purificação , Conotoxinas/metabolismo , Descoberta de Drogas/métodos , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Proteômica/métodos
17.
Toxins (Basel) ; 10(10)2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30347838

RESUMO

Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HN), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype (e.g., BoNT/A1). Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.


Assuntos
Toxinas Botulínicas/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Toxinas Botulínicas/química , Humanos , Neurotoxinas/química , Ligação Proteica , Domínios Proteicos
18.
Mar Drugs ; 16(11)2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30366463

RESUMO

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis, which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation.


Assuntos
Venenos de Cnidários/química , Venenos de Cnidários/genética , Toxinas Marinhas/química , Neurotoxinas/química , Neurotoxinas/genética , Anêmonas-do-Mar/química , Anêmonas-do-Mar/genética , Animais , Venenos de Cnidários/isolamento & purificação , Venenos de Cnidários/farmacologia , Mineração de Dados , Toxinas Marinhas/genética , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/farmacologia , Neurotoxinas/isolamento & purificação , Neurotoxinas/farmacologia , RNA/química , RNA/genética , Análise de Sequência de RNA , Pesquisa Médica Translacional
19.
ACS Chem Biol ; 13(11): 3107-3114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30296060

RESUMO

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.


Assuntos
Família Multigênica , Neurotoxinas/classificação , Neurotoxinas/genética , Saxitoxina/classificação , Saxitoxina/genética , Cianobactérias/genética , Dioxigenases/genética , Genes Bacterianos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neurotoxinas/química , Fosfoadenosina Fosfossulfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Ligação Proteica , Saxitoxina/análogos & derivados , Saxitoxina/síntese química , Saxitoxina/química , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismo , Transposases/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-30290365

RESUMO

The pond wolf spider, Pardosa pseudoannulata, is one of the most dominant predators of several agriculture insect pests including mainly the rice planthoppers and leafhoppers in paddy fields in Asia. Venom has been an attractive subject as the powerful weapon of spiders due to the complex components secreted from venom gland. The transcriptome of P. pseudoannulata venom gland was sequenced in order to understand the diversity of venom components at the molecular level and provide a new perspective for insect pest management. A total of 48 potential peptide toxins were identified from 75,980 assembled transcripts. Among them, 32 putative neurotoxin precursors were classified into six families systematically. In addition to neurotoxins, peptide toxins with potential antiviral and antifungal activities were annotated as well as the protease inhibitors. The diversity of P. pseudoannulata venom was firmly demonstrated with the presence of astacin-like metalloprotease toxins, Kunitz-type serine protease inhibitors, venom allergen 5, hyaluronidase and other important venom components. In addition, the tissue expression profiles of the toxin-related genes were investigated in venom gland, brain, and fat body. The present study classified the diverse venom components P. pseudoannulata and provided essential information of potential insecticidal toxins for the biological control of insect pests.


Assuntos
Venenos de Aranha/genética , Aranhas/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Insetos/fisiologia , Neurotoxinas/química , Neurotoxinas/genética , Oryza/parasitologia , Peptídeos/química , Peptídeos/genética , Filogenia , Comportamento Predatório , Alinhamento de Sequência , Venenos de Aranha/química , Aranhas/química , Aranhas/fisiologia
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