RESUMEN
PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H+-ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.
Asunto(s)
Proteínas de Insectos/genética , Insecticidas/toxicidad , Péptidos/toxicidad , Pisum sativum/genética , Proteínas de Plantas/toxicidad , Toxinas Biológicas/toxicidad , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Apoptosis , Caspasa 3/genética , Caspasa 3/metabolismo , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/metabolismo , Regulación de la Expresión Génica , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Insecticidas/aislamiento & purificación , Insecticidas/metabolismo , Pisum sativum/química , Pisum sativum/parasitología , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Unión Proteica , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Semillas/química , Semillas/genética , Semillas/parasitología , Toxinas Biológicas/aislamiento & purificación , Toxinas Biológicas/metabolismo , Tribolium/efectos de los fármacos , Tribolium/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Gorgojos/efectos de los fármacos , Gorgojos/metabolismoRESUMEN
The Pea Albumin 1 subunit b (PA1b) peptide is an entomotoxin extracted from legume seeds with lethal activity towards several insect pests. Its toxic activity occurs after the perception of PA1b by a plasmalemmic proton pump (V-ATPase) in the insects. Assays revealed that PA1b showed no activity towards mammalian cells displaying high V-ATPase activity. Similarly, PA1b displayed no binding activity and no biological activity towards other non-insect organisms. We demonstrate here that binding to labelled PA1b was found in all the insect families tested, regardless of the sensitivity or insensitivity of the individual species. The coleopteran Bruchidae, which are mainly legume seed pests, were found to be fully resistant. A number of insect species were seen to be insensitive to the toxin although they exhibited binding activity for the labelled PA1b. The fruit fly, Drosophila melanogaster (Diptera), was generally insensitive when maintained on an agar diet, but the fly appeared to be sensitive to PA1b in bioassays using a different diet. In conclusion, the PA1b toxin provides legumes with a major source of resistance to insects, and insects feeding on legume seeds need to overcome this plant resistance by disrupting the PA1b - V-ATPase interaction.
Asunto(s)
Fabaceae/química , Insectos/efectos de los fármacos , Plaguicidas/toxicidad , Proteínas de Plantas/toxicidad , ATPasas de Translocación de Protón Vacuolares/química , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Escarabajos/efectos de los fármacos , Drosophila melanogaster/efectos de los fármacos , Humanos , Proteínas de Insectos/química , Resistencia a los Insecticidas , Células MCF-7 , Ratones , Datos de Secuencia Molecular , Osteoclastos/efectos de los fármacos , Plaguicidas/química , Proteínas de Plantas/química , Semillas/química , Pruebas de ToxicidadRESUMEN
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.
Asunto(s)
Albúminas/metabolismo , Insecticidas/metabolismo , Pisum sativum/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Albúminas/antagonistas & inhibidores , Secuencia de Bases , Benzofenonas/metabolismo , Sitios de Unión , Biotina/metabolismo , Cartilla de ADN , Insecticidas/química , Microscopía Electrónica , Etiquetas de Fotoafinidad , Unión Proteica , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
The PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin extract from Legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils. This 37 amino-acid cysteine-rich peptide has been, until now, obtained by biochemical purification or chemical synthesis. In this paper, we present our results for the transient production of the peptide in Nicotiana benthamiana by agro-infiltration, with a yield of about 35 µg/g of fresh leaves and maximum production 8 days after infiltration. PA1b is part of the PA1 gene which, after post-translational modifications, encodes two peptides (PA1b and PA1a). We show that transforming tobacco with the PA1b cDNA alone does not result in production of the toxin and, in fact, the entire cDNA is necessary, raising the question of the role of PA1a. We constructed a PA1-cassette, allowing for the quick "cut/paste" of different PA1b mutants within a conserved PA1 cDNA. This cassette enabled us to produce the six isoforms of PA1b which exist in pea seeds. Biological tests revealed that all the isoforms display similar activity, with the exception of one which is inactive. The lack of activity in this isoform led us to conclude that the amphiphilic nature of the peptide is necessary for activity. The possible applications of this expression system for other cysteine-rich biomolecules are discussed.
Asunto(s)
Insecticidas/química , Nicotiana/genética , Pisum sativum/química , Proteínas de Plantas/química , Subunidades de Proteína/química , Toxinas Biológicas/química , Secuencia de Aminoácidos , Agentes de Control Biológico , ADN Complementario , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Insecticidas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Pisum sativum/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Nicotiana/metabolismo , Toxinas Biológicas/biosíntesis , Toxinas Biológicas/genéticaRESUMEN
BACKGROUND: Because of the increasingly concern of consumers and public policy about problems for environment and for public health due to chemical pesticides, the search for molecules more safe is currently of great importance. Particularly, plants are able to fight the pathogens as insects, bacteria or fungi; so that plants could represent a valuable source of new molecules. RESULTS: It was observed that Medicago truncatula seed flour displayed a strong toxic activity towards the adults of the rice weevil Sitophilus oryzae (Coleoptera), a major pest of stored cereals. The molecule responsible for toxicity was purified, by solvent extraction and HPLC, and identified as a saponin, namely 3-GlcA-28-AraRhaxyl-medicagenate. Saponins are detergents, and the CMC of this molecule was found to be 0.65 mg per mL. Neither the worm Caenorhabditis elegans nor the bacteria E. coli were found to be sensitive to this saponin, but growth of the yeast Saccharomyces cerevisiae was inhibited at concentrations higher than 100 µg per mL. The purified molecule is toxic for the adults of the rice weevils at concentrations down to 100 µg per g of food, but this does not apply to the others insects tested, including the coleopteran Tribolium castaneum and the Sf9 insect cultured cells. CONCLUSIONS: This specificity for the weevil led us to investigate this saponin potential for pest control and to propose the hypothesis that this saponin has a specific mode of action, rather than acting via its non-specific detergent properties.
RESUMEN
PA1b (for pea albumin 1 subunit b) is a plant bioinsecticide lethal to several pests that are important in agriculture or human health. PA1b belongs to the inhibitory cystine knot family or knottin family. Originating from a plant (the garden pea) commonly eaten by humans without any known toxic or allergic effects, PA1b is a candidate for transgenic applications and is one of the most promising biopesticides for pest control. Using whole-cell patch-clamp techniques on Sf9 PA1b-sensitive lepidopteran insect cells, we discovered that PA1b reversibly blocked ramp membrane currents in a dose-dependent manner (EC(50) = 0.52 µM). PA1b had the same effect as bafilomycin, a specific inhibitor of the vacuolar proton pump (V-type H(+)-ATPase), and the PA1b-sensitive current depended on the internal proton concentration. Biochemical assays on purified V-ATPase from the lepidopteran model Manduca sexta showed that PA1b inhibited the V(1)V(0)-type H(+)-ATPase holoenzyme activity (IC(50) â¼ 70 nM) by interacting with the membrane-bound V(0) part of the V-ATPase. V-ATPase is a complex protein that has been studied increasingly because of its numerous physiological roles. In the midgut of insects, V-ATPase activity is essential for energizing nutrient absorption, and the results reported in this work explain the entomotoxic properties of PA1b. Targeting V-ATPase is a promising means of combating insect pests, and PA1b represents the first peptidic V-ATPase inhibitor. The search for V-ATPase inhibitors is currently of great importance because it has been demonstrated that V-ATPase plays a role in so many physiological processes.
Asunto(s)
Miniproteínas Nodales de Cistina/farmacología , Proteínas de Insectos/antagonistas & inhibidores , Insecticidas/farmacología , Manduca/enzimología , Proteínas de Plantas/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Línea Celular , Humanos , Spodoptera/enzimologíaRESUMEN
PA1b (Pea Albumin 1, subunit b) is a peptide extract from pea seeds showing significant insecticidal activity against certain insects, such as cereal weevils (genus Sitophilus), the mosquitoes Culex pipiens and Aedes aegyptii, and certain species of aphids. PA1b has great potential for use on an industrial scale and for use in organic farming: it is extracted from a common plant; it is a peptide (and therefore suitable for transgenic applications); it can withstand many steps of extraction and purification without losing its activity; and it is present in a seed regularly consumed by humans and mammals without any known toxicity or allergenicity. The potential of this peptide to limit pest damage has stimulated research concerning its host range, its mechanism of action, its three-dimensional structure, the natural diversity of PA1b and its structure-function relationships.