RESUMEN
Dufour's gland is associated with the venom apparatuses of social wasps and bees. This location and its evolutionary adaptations indicate that it could be involved in the production of alarm pheromones in the social wasp Polybia paulista. To investigate this hypothesis, the volatile composition of this gland was analyzed and compared to that in the venom. Eighteen compounds were identified as secreted by Dufour's gland, and 16 of these compounds were also identified in the venom, suggesting that the compounds produced by the gland are secreted and mixed with venom in the venom reservoir of this wasp. These compounds were subjected to a field bioassay to investigate their potential action as alarm pheromones. Alcohols and aldehydes elicited the alert behavior in workers, luring them outside the nest, whereas acids attracted the workers in the direction of the source; fatty acid methyl esters elicited aggression. These results suggest that Dufour's gland produces alarm pheromones. To corroborate this hypothesis the proteomic complement of this gland was assigned using a shot-gun strategy; 59 proteins were identified, and the results indicate specialization of Dufour's gland for the metabolism of fatty acids (elongation, esterification unsaturation, reduction, and decarboxylation) in the biosynthesis of alarm pheromones. BIOLOGICAL SIGNIFICANCE: The present knowledge about the role of Dufour's gland among aculeate Hymenoptera insects suggests that it may have many different roles related to the biosynthesis and secretion of chemical markers for different biological functions, though none are related to the elicitation of alarm behaviors for coordinating a mass attack of the colony against intruders. The present study combined the analysis of secreted volatile compounds (as metabolites) with proteome assignments and a field bioassay with synthetic compounds to clearly demonstrate that Dufour's gland does in fact biosynthesize alarm pheromones in social wasps. This strategy may be reproduced in other investigations related to pheromone production in other insects.
Asunto(s)
Metabolómica/métodos , Feromonas/química , Proteómica/métodos , Glándulas Odoríferas/química , Venenos de Avispas/química , Animales , Conducta Animal , Proteínas de Insectos/análisis , Proteínas de Insectos/metabolismo , AvispasRESUMEN
Fire ants are well-known by their aggressive stinging behavior, causing many stinging incidents of medical importance. The limited availability of fire ant venom for scientific and clinical uses has restricted, up to now, the knowledge about the biochemistry, immunology, and pharmacology of these venoms. For this study, S. invicta venom was obtained commercially and used for proteomic characterization. For this purpose, the combination of gel-based and gel-free proteomic strategies was used to assign the proteomic profile of the venom from the fire ant S. invicta. This experimental approach permitted the identification of 46 proteins, which were organized into four different groups according to their potential role in fire ant venom: true venom components, housekeeping proteins, body muscle proteins, and proteins involved in chemical communication. The active venom components that may not present toxic roles were classified into three subgroups according to their potential functions: self-venom protection, colony asepsis, and chemical communication. Meanwhile, the proteins classified as true toxins, based on their functions after being injected into the victims' bodies by the fire ants, were classified in five other subgroups: proteins influencing the homeostasis of the victims, neurotoxins, proteins that promote venom diffusion, proteins that cause tissue damage/inflammation, and allergens.
Asunto(s)
Venenos de Hormiga/química , Hormigas/química , Proteínas de Insectos/análisis , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Hormigas/metabolismo , Electroforesis en Gel Bidimensional , Proteínas de Insectos/química , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico , Proteoma/química , ProteómicaRESUMEN
It is well known that the activation of mast cells due to the binding of mastoparan to the G(α) subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca(2+) -independent FcεRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca(2+) -dependent FcεRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.
Asunto(s)
Endosomas/metabolismo , Proteínas de Unión al GTP/metabolismo , Mastocitos/metabolismo , Péptidos/metabolismo , Proteómica , Venenos de Avispas/metabolismo , Secuencia de Aminoácidos , Animales , Degranulación de la Célula , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endosomas/enzimología , Exocitosis , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular , Masculino , Espectrometría de Masas , Mastocitos/citología , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Ratas , Venenos de Avispas/síntesis química , Venenos de Avispas/química , Avispas/químicaRESUMEN
Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/ß-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.
Asunto(s)
Ácidos Grasos/metabolismo , Modelos Moleculares , PPAR gamma/química , PPAR gamma/metabolismo , Conformación Proteica , Tiazolidinedionas/metabolismo , Células 3T3 , Animales , Compuestos Azo , Cristalización , Ácidos Grasos/farmacología , Células HeLa , Humanos , Ratones , Simulación de Dinámica Molecular , PPAR gamma/agonistas , Estructura Terciaria de ProteínaRESUMEN
The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the ß-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2', H3, and the ß-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators.
Asunto(s)
Luteolina/farmacología , PPAR gamma/agonistas , Células 3T3-L1 , Animales , Secuencia de Bases , Cartilla de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Luteolina/química , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Ácido Mirístico/química , PPAR gamma/química , PPAR gamma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Tiazolidinedionas/antagonistas & inhibidores , Tiazolidinedionas/farmacologíaRESUMEN
The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARγ and RXRα is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARγ alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARγ remains in the monomeric form by itself but forms heterodimers with hRXRα. The low-resolution models of hPPARγ/RXRα complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.
Asunto(s)
Modelos Moleculares , PPAR gamma/química , Secuencia de Aminoácidos , Cromatografía en Gel , ADN/metabolismo , Medición de Intercambio de Deuterio , Humanos , Hidrodinámica , Espectrometría de Masas , Datos de Secuencia Molecular , PPAR gamma/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/metabolismo , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
When searching for prospective novel peptides, it is difficult to determine the biological activity of a peptide based only on its sequence. The "trial and error" approach is generally laborious, expensive and time consuming due to the large number of different experimental setups required to cover a reasonable number of biological assays. To simulate a virtual model for Hymenoptera insects, 166 peptides were selected from the venoms and hemolymphs of wasps, bees and ants and applied to a mathematical model of multivariate analysis, with nine different chemometric components: GRAVY, aliphaticity index, number of disulfide bonds, total residues, net charge, pI value, Boman index, percentage of alpha helix, and flexibility prediction. Principal component analysis (PCA) with non-linear iterative projections by alternating least-squares (NIPALS) algorithm was performed, without including any information about the biological activity of the peptides. This analysis permitted the grouping of peptides in a way that strongly correlated to the biological function of the peptides. Six different groupings were observed, which seemed to correspond to the following groups: chemotactic peptides, mastoparans, tachykinins, kinins, antibiotic peptides, and a group of long peptides with one or two disulfide bonds and with biological activities that are not yet clearly defined. The partial overlap between the mastoparans group and the chemotactic peptides, tachykinins, kinins and antibiotic peptides in the PCA score plot may be used to explain the frequent reports in the literature about the multifunctionality of some of these peptides. The mathematical model used in the present investigation can be used to predict the biological activities of novel peptides in this system, and it may also be easily applied to other biological systems.
Asunto(s)
Venenos de Artrópodos/química , Productos Biológicos/química , Defensinas/química , Hemolinfa/química , Himenópteros/química , Péptidos/química , Algoritmos , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , Punto Isoeléctrico , Modelos Teóricos , Análisis de Componente Principal , Estructura Secundaria de ProteínaRESUMEN
Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-terminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILGTILGLLKGL-NH(2)) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH(2)) only presents chemotaxis for PMNL. However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration.
Asunto(s)
Factores Quimiotácticos/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Venenos de Avispas/química , Avispas/fisiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Degranulación de la Célula/efectos de los fármacos , Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Dicroismo Circular/métodos , Hemólisis/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Pruebas de Sensibilidad Microbiana , Neutrófilos/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Análisis de Secuencia de Proteína , Espectrometría de Masa por Ionización de Electrospray , Venenos de Avispas/farmacologíaRESUMEN
Polybioside (1) was isolated from the venom of the social wasp Polybia paulista, and its structure was assigned as 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl 3-(1H-imidazol-4-yl)propanimidate by NMR and MS protocols. The application of polybioside in rat brain, followed by the detection of c-Fos protein expression in some brain regions, indicated the compound is neuroactive in a number of brain areas. Polybioside causes convulsions in rats, even when peripherally applied.
Asunto(s)
Fármacos del Sistema Nervioso Central/aislamiento & purificación , Fármacos del Sistema Nervioso Central/farmacología , Glucósidos/aislamiento & purificación , Glucósidos/farmacología , Imidazoles/aislamiento & purificación , Imidazoles/farmacología , Venenos de Avispas/química , Avispas/química , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Brasil , Fármacos del Sistema Nervioso Central/química , Glucósidos/química , Imidazoles/química , Masculino , Estructura Molecular , Neuronas/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Wistar , Convulsiones/inducido químicamenteRESUMEN
Colonial spiders evolved a differential prey-capture behaviour in concert with their venom chemistry, which may be a source of novel drugs. Some highly active tetrahydro-beta-carboline (THbetaC) toxins were recently isolated from the venom of the colonial spider Parawixia bistriata; the spiders use these toxins as part of their chemical arsenal to kill and/or paralyze preys. The major THbetaC compound isolated from this venom was identified as 6-hydroxytrypargine, also known as PwTX-I. Most natural compounds of animal origin occur in low abundance, and the natural abundance of PwTX-I is insufficient for complete functional characterization. Thus, PwTx-I was synthesized using a Pictet-Spengler condensation strategy, and the stereoisomers of the synthetic toxin were separated by chiral chromatography. The fraction of venom containing a mixture of three natural THbetaC toxins and enantiomers of PwTx-I were analyzed for inhibition of monoamine oxidase (MAO)-A and -B and for toxicity to insects. We reveal that the mixture of the natural THbetaC toxins, as well as the enantiomers of PwTx-I, were non-competitive inhibitors of MAO-A and MAO-B and caused potent paralysis of honeybees. The (-)-PwTX-I enantiomer is 2-fold more potent than the (+)-PwTX-I enantiomer in the assays performed.
Asunto(s)
Alcaloides Indólicos/toxicidad , Inhibidores de la Monoaminooxidasa/farmacología , Venenos de Araña/química , Animales , Alcaloides Indólicos/aislamiento & purificación , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Monoaminooxidasa/efectos de los fármacos , Monoaminooxidasa/metabolismo , Espectrometría de Fluorescencia , Arañas , EstereoisomerismoRESUMEN
The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.
Asunto(s)
Proteínas de Unión al ADN/química , Receptores Citoplasmáticos y Nucleares/química , Receptores de Esteroides/química , Factores de Transcripción/química , Dicroismo Circular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Dimerización , Espectrometría de Masas , Modelos Biológicos , Modelos Moleculares , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Estructura Secundaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Receptores de Glucocorticoides/química , Receptores de Esteroides/genética , Receptores de Esteroides/aislamiento & purificación , Dispersión del Ángulo Pequeño , Soluciones , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and 1H NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12+/-3 etag/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat).