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

A Rhodnius prolixus Insulin Receptor and Its Conserved Intracellular Signaling Pathway and Regulation of Metabolism

The insulin signaling pathway is a modulator of metabolism in insects and can regulate functions associated with growth and development, as well as lipid and carbohydrate balance. We have previously reported the presence of an insulin-like peptide and an insulin-like growth factor in Rhodnius prolixus, which are involved in the homeostasis of lipids and carbohydrates in post-feeding and non-feeding periods. In the present study, we have characterized the first insulin receptor (IR) to be discovered in R. prolixus, Rhopr-IR, and investigated its intracellular signaling cascade and its role in nutrient control. We identified a candidate protein sequence within R. prolixus putative peptidome and predicted its conserved features using bioinformatics. Tissue-specific expression analyses indicated that the Rhopr-IR transcript is differentially-expressed in all tissues tested, with the highest values observed in the central nervous system (CNS). Treatment of insects with the IR kinase activator BpV(phen), glucose, or porcine insulin resulted in the activation of protein phosphorylation in the fat body, and stimulated the phosphorylation of protein kinase Akt, an evolutionarily conserved key regulator of the intracellular insulin signaling cascade. We also observed activation of Akt and phosphorylation of its downstream targets glycogen synthase kinase 3 β (GSK3β) and the transcription factor FOXO for several days following a blood meal. We used dsRNA to knockdown transcript expression and examined the resulting effects on metabolism and intracellular signaling. Furthermore, knockdown of the Rhopr-IR transcript increased lipid levels in the hemolymph, while reducing lipid content in the fat body. Interestingly, the levels of carbohydrates in the hemolymph and in the fat body did not show any alterations. The activation of Akt and phosphorylation of FOXO were also reduced in knockdown insects, while the phosphorylation pattern of GSK3β did not change. Our results support the identification of the first IR in R. prolixus and suggest that Rhopr-IR signaling is involved in hemolymph nutrient homeostasis and fat body storage both in post-feeding and in non-feeding stages. These metabolic effects are likely regulated by the activation of Akt and downstream cascades similar to mammalian insulin signaling pathways

Effect of the urease-derived peptide Jaburetox on the central nervous system of Triatoma infestans (Insecta: Heteroptera)

Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species.Fil: Galvani, Gerónimo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales; Argentina. Universidad Austral; ArgentinaFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; Argentina. Universidad Nacional de Cordoba. Facultad de Ciencias Quimicas; ArgentinaFil: Coronel, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Nowicki, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Demartini, Diogo R.. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Postal, Melissa F.. Universidade Federal 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; ArgentinaFil: Carlini, Celia. Universidade Federal do Rio Grande do Sul; BrasilFil: Settembrini, Beatriz Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales; Argentina. Universidad Austral; Argentin

Soybean ubiquitous urease with purification facilitator: An addition to the moonlighting studies toolbox

Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. In soybean (Glycine max), the embryo-specific urease (eSBU), the ubiquitous urease (uSBU), and a third isoform (SBU-III) are synthesized. Our group has previously demonstrated that eSBU, purified from seeds, has antifungal properties against phytopathogenic fungi, entomotoxicity against Dysdercus peruvianus, the ability to induce blood platelet aggregation, and these properties are independent of its enzymatic activity. Here we describe the biological properties of apo-uSBU fused to glutathione S-transferase (GST) produced in Escherichia coli. Removal of GST affected apo-uSBU stability. We performed a Response Surface Methodology to optimize GST-uSBU production to 5 mg per liter and then bioassays were carried out. The recombinant protein exhibited inhibitory effects on filamentous fungi and affected fungal secondary metabolism. Candida albicans and C. tropicalis were also susceptible to GST-uSBU and formed pseudo-hyphae. The fusion protein was toxic against Rhodnius prolixus, with the toxicity being accompanied by in vivo and in vitro hemocyte aggregation. Rabbit platelet also aggregated in the presence of GST-uSBU. Thus, uSBU displayed similar biological properties as previously described for eSBU even when fused to GST, reinforcing the proposed role of ureases in plant defense.Fil: Martinelli, Anne H.S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Lopes, Fernanda C.. Universidade Federal do Rio Grande do Sul; BrasilFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. University of Toronto; CanadáFil: Ligabue-Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Kappaun, Karine. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Tichota, Deise M.. Universidade Federal do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. 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: Moyetta, Natalia Rita. 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: Demartini, Diogo R.. Universidade Federal do Rio Grande do Sul; BrasilFil: Postal, Melissa. Universidade Federal do Rio Grande do Sul; BrasilFil: Medeiros-Silva, Monica. Universidade Federal do Rio Grande do Sul; BrasilFil: Becker-Ritt, Arlete Beatriz. Universidade Luterana Do Brasil; BrasilFil: Pasquali, Giancarlo. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; Brasi