Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Peptide diversity in the venom of the social wasp Polybia paulista (Hymenoptera): A comparison of the intra- and inter-colony compositions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

T-type calcium channel:from physiological function to therapeutical targeting – Exploring neuronal development

Tämän tutkimuksen tarkoitus oli tutkia T-tyypin kalsiumkanavan toimintaa ja sen mahdollista roolia neuronaalisten kantasolujen migraatiossa. T-tyypin kalsiumkanavan tehtävän kehittyneissä aivoissa tiedetään olevan elektroenkefalografisten oskillaatioiden tuottaminen. Nämä taas ovat eräiden fysiologisten ja patofysiologisten tapahtumien säätelyssä avainasemassa. Tällaisia tapahtumia ovat uni, muisti, oppiminen ja epileptiset poissaolokohtaukset. Näiden lisäksi T-tyypin kalsiumkanavalla on myös periferaalisia vaikutuksia, mutta tämä tutkielma keskittyy sen neuronaalisiin toimintoihin. Tämän matalan jännitteen säätelemän kanavan toiminta neurogeneesin aikana on vähemmän tutkittua ja tunnettua kuin sen vaikutukset kehittyneissä aivoissa. T-tyypin kalsiumkanavan tiedetään edistävän kantasolujen proliferaatiota ja erilaistumista neurogeneesiksen aikana, mutta vaikutukset niiden migraatioon ovat vähemmän tunnetut. Tämä tutkimus näyttää T-tyypin kalsiumkanavan todennäköisesti osallistuvan neuronaaliseen migraatioon hiiren alkion subventrikkeli alueelta eristetyillä kanta- tai progeniittorisoluilla tehdyissä kokeissa. Selektiiviset T-tyypin kalsiumkanavan antagonistit, etosuksimidi, nikkeli ja skorpionitoksiini, kurtoxin hidastivat migraatiota erilaistuvissa progeniittorisoluissa. Tämä tutkimus koostuu kirjallisuuskatsauksesta ja kokeellisesta osasta. Tämän tutkimuksen toinen tarkoitus oli esitellä vaihtoehtoinen lähestymistapa invasiiviselle kantasoluterapialle, joka vaatii kantasolujen viljelyä ja siirtämistä ihmiseen. Tämä toinen tapa on endogeenisten kantasolujen eiinvasiivinen stimulointi, jolla ne saadaan migratoitumaan kohdekudokseen, erilaistumaan siellä ja tehtävänsä suoritettuaan lopettamaan jakaantumisen. Non-invasiivinen kantasoluterapia on vasta tiensä alussa, ja tarvitsee farmakologista osaamista kehittyäkseen. Joitain onnistuneita ei-invasiivisia hoitoja on jo tehty selkärangan vaurioiden korjaamisessa. Vastaavanlaisia menetelmiä voitaisiin käyttää myös keskushermoston vaurioiden ja neurodegeneratiivisten sairauksien hoidossa. Näiden menetelmien kehittäminen vaatii endogeenisten kantasoluja inhiboivien ja indusoivien mekanismien tuntemista. Yksi tärkeä kantasolujen erilaistumista stimuloiva tekijä on kalsiumioni. Jänniteherkät kalsiumkanavat osallistuvat kaikkiin neurogeneesiksen eri vaiheisiin. T-tyypin kalsiumkanava, joka ekspressoituu suuressa määrin keskushermoston kehityksen alkuvaiheessa ja vähenee neuronaalisen kehityksen edetessä, saattaa olla oleellisessa asemassa progeniittorisolujen ohjaamisessa.

Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins

Disulfide crosslinks are ubiquitous in natural peptides and proteins, providing rigidity to polypeptide scaffolds. The assignment of disulfide connectivity in multiple crosslinked systems is often difficult to achieve. Here, we show that rapid unambiguous characterisation of disulfide connectivity can be achieved through direct mass spectrometric CID fragmentation of the disulfide intact polypeptides. The method requires a direct mass spectrometric fragmentation of the native disulfide bonded polypeptides and subsequent analysis using a newly developed program, DisConnect. Technical difficulties involving direct fragmentation of proteins are surmounted by an initial proteolytic nick and subsequent determination of the structures of these proteolytic peptides through DisConnect. While the connectivity in proteolytic fragments containing one cystine is evident from the MS profile alone, those with multiple cystines are subjected to subsequent mass spectrometric fragmentation. The wide applicability of this method is illustrated using examples of peptide hormones, peptide toxins, proteins, and disulfide foldamers of a synthetic analogue of a marine peptide toxin. The method, coupled with DisConnect, provides an unambiguous, straightforward approach, especially useful for the rapid screening of the disulfide crosslink fidelity in recombinant proteins, determination of disulfide linkages in natural peptide toxins and characterization of folding intermediates encountered in oxidative folding pathways.

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of blue-green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue-green algae meal. Diet 1 was used as control with no blue-green algae meal whereas the content in diets 2-6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow-through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3-6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish-fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish-fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).

Functional and Structural Diversification of the Anguimorpha Lizard Venom System

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this similar to 130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding do-mains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A 2 toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms. Molecular & Cellular Proteomics 9:2369-2390, 2010.

Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Modulation of insect Cav channels by peptidic spider toxins

Insects have a much smaller repertoire of voltage-gated calcium (Ca-v) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(v)1, Ca(v)2, and Ca(v)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca-v channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca-v channels, since severe loss-of-function mutations in genes encoding the pore-forming a, subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca-v channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca-v channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca-v channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca-v channels. This review focuses on peptidic spider toxins that specifically target insect Ca-v channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests. (c) 2006 Elsevier Ltd. All rights reserved.

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

Structure-function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background: The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised - with an intra-molecular disulphide bridge; and reduced - in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now. Methods: Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge. Results: Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway. Conclusion: The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine. General significance: The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies. © 2013 Elsevier B.V.

Functional and Topological Studies with Trp-Containing Analogs of the Peptide StII(1-30) Derived From the N-Terminus of the Pore Forming Toxin Sticholysin II: Contribution to Understand its Orientation in Membrane

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)