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1.
Adv Exp Med Biol ; 1131: 27-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646506

RESUMO

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.


Assuntos
Bioensaio , Canais Iônicos , Receptores Acoplados a Proteínas-G , Animais , Bioensaio/tendências , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Humanos , Canais Iônicos/análise , Receptores Acoplados a Proteínas-G/análise
2.
Mar Drugs ; 17(8)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344776

RESUMO

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

3.
J Physiol ; 597(14): 3751-3768, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087362

RESUMO

KEY POINTS: Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The ß-scorpion toxin, Cn2, is selective for NaV 1.6 in dorsal root ganglion neurons. NaV 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. NaV 1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted NaV isoforms in pain generation. ABSTRACT: Peripheral sensory neurons express multiple voltage-gated sodium channels (NaV ) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): NaV 1.7, NaV 1.8 and NaV 1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests NaV 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV 1.6 channels in peripheral sensory neurons. The potent and NaV 1.6 selective ß-scorpion toxin Cn2 was used to assess the effect of NaV 1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV 1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, NaV 1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on NaV 1.6 channels in sensory neurons. Activation of NaV 1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing NaV 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.

4.
J Biol Chem ; 294(22): 8745-8759, 2019 May 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.

5.
Elife ; 82019 Feb 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.

6.
Toxins (Basel) ; 10(12)2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30513724

RESUMO

Cone snails (genus Conus) are venomous marine snails that inject prey with a lethal cocktail of conotoxins, small, secreted, and cysteine-rich peptides. Given the diversity and often high affinity for their molecular targets, consisting of ion channels, receptors or transporters, many conotoxins have become invaluable pharmacological probes, drug leads, and therapeutics. Transcriptome sequencing of Conus venom glands followed by de novo assembly and homology-based toxin identification and annotation is currently the state-of-the-art for discovery of new conotoxins. However, homology-based search techniques, by definition, can only detect novel toxins that are homologous to previously reported conotoxins. To overcome these obstacles for discovery, we have created ConusPipe, a machine learning tool that utilizes prominent chemical characters of conotoxins to predict whether a certain transcript in a Conus transcriptome, which has no otherwise detectable homologs in current reference databases, is a putative conotoxin. By using ConusPipe on RNASeq data of 10 species, we report 5148 new putative conotoxin transcripts that have no homologues in current reference databases. 896 of these were identified by at least three out of four models used. These data significantly expand current publicly available conotoxin datasets and our approach provides a new computational avenue for the discovery of novel toxin families.

7.
Toxins (Basel) ; 10(11)2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30400621

RESUMO

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 µg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/µL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/ß defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

8.
Toxicon ; 154: 15-27, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30267720

RESUMO

The insects are a hyperdiverse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.

9.
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.

10.
Sci Adv ; 4(9): eaau4640, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30214940

RESUMO

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa. We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX1-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX1-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.

11.
Expert Rev Proteomics ; 14(10): 931-939, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28879805

RESUMO

INTRODUCTION: Animal venoms are complex chemical arsenals. Most venoms are rich in bioactive peptides with proven potential as research tools, drug leads and drugs. Areas covered: We review recent advances in venom-peptide discovery, particularly the adoption of combined transcriptomic/proteomic approaches for the exploration of venom composition. Expert commentary: Advances in transcriptomics and proteomics have dramatically altered the manner and rate of venom-peptide discovery. The increasing trend towards a toxin-driven approach, as opposed to traditional target-based screening of venoms, is likely to expedite the discovery of venom-peptides with novel structures and new and unanticipated mechanisms of action. At the same time, these advances will drive the development of higher-throughput approaches for target identification. Taken together, these approaches should enhance our understanding of the natural ecological function of venom peptides and increase the rate of identification of novel venom-derived pharmacological tools, drug leads and drugs.


Assuntos
Descoberta de Drogas/métodos , Peptídeos/química , Proteômica/métodos , Peçonhas/química , Animais , Humanos , Peptídeos/uso terapêutico , Peçonhas/uso terapêutico
12.
Neuropharmacology ; 127: 79-86, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28689026

RESUMO

Diabetes mellitus is a chronic disease caused by a deficiency in production of insulin by the beta cells of the pancreas (type 1 diabetes, T1D), or by partial deficiency of insulin production and the ineffectiveness of the insulin produced (type 2 diabetes, T2D). Animal venoms are a unique source of compounds targeting ion channels and receptors in the nervous and cardiovascular systems. In recent years, several venom peptides have also emerged as pharmacological tools and therapeutics for T1D and T2D. Some of these peptides act directly as mimics of endogenous metabolic hormones while others act on ion channels expressed in pancreatic beta cells. Here, we provide an overview of the discovery of these venom peptides, their mechanisms of action in the context of diabetes, and their therapeutic potential for the treatment of this disease. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'


Assuntos
Diabetes Mellitus/tratamento farmacológico , Peptídeos/uso terapêutico , Peçonhas/química , Peçonhas/uso terapêutico , Animais , Humanos , Peptídeos/farmacologia , Peçonhas/farmacologia
13.
Mar Drugs ; 15(5)2017 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-28531118

RESUMO

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.


Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Caramujo Conus/fisiologia , Venenos de Moluscos/química , Sequência de Aminoácidos , Animais , Venenos de Moluscos/metabolismo , Transcriptoma
14.
Biochemistry ; 56(19): 2455-2466, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28437072

RESUMO

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed ß-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11-22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc11-22[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc11-22[NNN12-14] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 µM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.


Assuntos
Conotoxinas/química , Peptídeos Cíclicos/química , Engenharia de Proteínas , Proteínas Supressoras da Sinalização de Citocina/química , Conotoxinas/genética , Conotoxinas/metabolismo , Cisteína/química , Cistina/química , Epitopos , Humanos , Cinética , Isótopos de Nitrogênio , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Conformação Proteica em Folha beta , Dobramento de Proteína , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ressonância de Plasmônio de Superfície
15.
Toxicon ; 129: 113-122, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28216409

RESUMO

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.


Assuntos
Aminoácidos/análise , Venenos de Moluscos/química , Peptídeos Cíclicos/química , Animais , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Caramujos/química , Canais de Cátion TRPC/metabolismo , Transcriptoma
16.
Gen Comp Endocrinol ; 244: 11-18, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-26301480

RESUMO

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey's nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins.


Assuntos
Peptídeos/metabolismo , Caramujos , Peçonhas/metabolismo , Animais , Conotoxinas
17.
Toxicon ; 123: 56-61, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27777069

RESUMO

The discovery of insulin and its use for the treatment of diabetes is undoubtedly one of the true successes of modern medicine. Injectable insulin would prove the first effective treatment for a previously incurable and usually fatal disease. Soon after however, the powerful effects of insulin overdose would be reported, and subsequently exploited for dubious medical and sometimes nefarious purposes. In this article we describe the discovery that certain venomous marine snails of the genus Conus also exploit the powerful effects of insulin overdose, employing it as a weapon for prey capture.


Assuntos
Caramujo Conus/fisiologia , Insulina/toxicidade , Venenos de Moluscos/toxicidade , Animais , Caramujo Conus/genética , Insulina/química , Venenos de Moluscos/química , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína
18.
Chem Commun (Camb) ; 52(24): 4446-9, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26892179

RESUMO

A facile stereoselective synthesis of cis and trans unsaturated dicarba peptides has been established using preformed diaminosuberic acid derivatives as bridging units. In addition, characteristic spectral differences in the (13)C-NMR spectra of the cis- and trans-isomers show that the chemical shift of carbons in the Δ4,5-diaminosuberic acid residue can be used to assign stereochemistry in unsaturated dicarba peptides formed from ring closing metathesis of linear peptide sequences.


Assuntos
Peptídeos/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Conformação Proteica , Estereoisomerismo
19.
Structure ; 24(2): 293-9, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26774129

RESUMO

Certain peptide folds, owing to a combination of intrinsic stability and resilience to amino acid substitutions, are particularly effective for the display of diverse functional groups. Such "privileged scaffolds" are valuable as starting points for the engineering of new bioactive molecules. We have identified a precursor peptide expressed in the venom gland of the marine snail Conus victoriae, which appears to belong to a hitherto undescribed class of molluscan neuropeptides. Mass spectrometry matching with the venom confirmed the complete mature peptide sequence as a 31-residue peptide with a single disulfide bond. Solution structure determination revealed a unique peptide fold that we have designated the single disulfide-directed ß hairpin (SDH). The SDH fold is highly resistant to thermal denaturation and forms the core of several other multiple disulfide-containing peptide folds, including the inhibitor cystine knot. This elementary fold may offer a valuable starting point for the design and engineering of new bioactive peptides.


Assuntos
Venenos de Moluscos/química , Neuropeptídeos/química , Peptídeos/química , Caramujos/química , Animais , Cisteína , Espectrometria de Massas , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína
20.
Front Cell Neurosci ; 9: 404, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26500501

RESUMO

NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 µM) but not high (50 µM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

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