DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from “classical” cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.Fil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Fruttero, Leonardo Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ligabue Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. University of Toronto; CanadáFil: Arrese, Estela L.. Oklahoma State University; Estados UnidosFil: Soulages, José L.. Oklahoma State University; Estados UnidosFil: Settembrini, Beatriz Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Patrón de desarrollo ovárico en hembras de Triatoma sordida ayunadas

Entre los vectores secundarios de Trypanosoma cruzi - agente causante de enfermedad de chagas - Triatoma sordida es considerada una especie de importancia epidemiológica debido a su presencia en peridomicilios y a la tendencia a invadir y formar grandes colonias domésticas.Fil: Rodríguez, C. Universidad Nacional de Córdoba Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas. Cátedra Introducción a la Biología; Argentina.Fil: Rodríguez, C. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Fil: Aguirre, S. Universidad Nacional de Córdoba. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología. Departamento de Bioquímica Clínica; Argentina.Fil: Aguirre, S. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Nattero, J. Universidad Nacional de Córdoba Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas. Cátedra .Introducción a la Biología; Argentina.Fil: Nattero, J. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Fil: Canavoso, L. Universidad Nacional de Córdoba. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología. Departamento de Bioquímica Clínica; Argentina.Fil: Canavoso, L. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Crocco, L. Universidad Nacional de Córdoba Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Biológicas y Tecnológicas. Cátedra .Introducción a la Biología; Argentina.Fil: Crocco, L. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Zoología, Ornitología, Entomología, Etologí

The in vivo treatment with the plant urease “Jack Bean Urease” impaired reproduction in females of Rhodnius prolixus (Hemiptera: Reduviidae)

Ureases are enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. In recent decades, it has been postulated that plant ureases are also defense proteins against phytophagous insect species with potential biotechnological application. Previous reports demonstrated that the injection of "Jack Bean Urease" (JBU), the major isoform of urease from the legume Canavalia ensiformis, into the hemocele of triatomine insects, resulted in several toxic effects including activation of the immune response. Although the insecticidal effect of JBU was described several years ago, many aspects of its mechanism of action as well as the target organs remains largely uncharacterized. In particular, the effects of JBU on the female reproductive system and the consequences of sublethal doses have not been studied.Para acceder a la videoconferencia completa, hacer clic en "Enlace externo".Sociedad Latinoamericana de Ecología de Vectore

Regulation of Energy Stores and Feeding by Neuronal and Peripheral CREB Activity in Drosophila

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores and increased sensitivity to starvation. Expression of DN-CREB in the fat body also reduced glycogen levels, while it did not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies did not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body caused an obese-like phenotype. Using a transgenic CRE-luciferase reporter, we further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and β-adrenergic signaling, in the fat body reduced CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved

Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes

Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

Functional gene analysis in malaria mosquitoes reveals molecules underpinning the trade-off between efficient reproduction and the antiparasitic response

An RGS-Containing Sorting Nexin Controls Drosophila Lifespan

The pursuit of eternal youth has existed for centuries and recent data indicate that fat-storing tissues control lifespan. In a D. melanogaster fat body insertional mutagenic enhancer trap screen designed to isolate genes that control longevity, we identified a regulator of G protein signaling (RGS) domain containing sorting nexin, termed snazarus (sorting nexin lazarus, snz). Flies with insertions into the 5′ UTR of snz live up to twice as long as controls. Transgenic expression of UAS-Snz from the snz Gal4 enhancer trap insertion, active in fat metabolic tissues, rescued lifespan extension. Further, the lifespan extension of snz mutants was independent of endosymbiont, e.g., Wolbachia, effects. Notably, old snz mutant flies remain active and fertile indicating that snz mutants have prolonged youthfulness, a goal of aging research. Since mammals have snz-related genes, it is possible that the functions of the snz family may be conserved to humans

Drosophila Lipophorin Receptors Mediate the Uptake of Neutral Lipids in Oocytes and Imaginal Disc Cells by an Endocytosis-Independent Mechanism

Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR–like proteins in this process