Plant Ureases and Related Peptides: Understanding Their Entomotoxic Properties

Recently, ureases were included in the arsenal of plant defense proteins, alongside many other proteins with biotechnological potential such as insecticides. Isoforms of Canavalia ensiformis urease (canatoxin—CNTX and jack bean urease—JBURE-I) are toxic to insects of different orders. This toxicity is due in part to the release of a 10 kDa peptide from the native protein, by cathepsin-like enzymes present in the insect digestive tract. The entomotoxic peptide, Jaburetox-2Ec, exhibits potent insecticidal activity against several insects, including many resistant to the native ureases. JBURE-I and Jaburetox-2Ec cause major alterations of post-feeding physiological processes in insects, which contribute to, or can be the cause of, their entomotoxic effect. An overview of the current knowledge on plant urease processing and mechanisms of action in insects is presented in this review

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

Structure-Function insights of Jaburetox and Soyuretox: Novel intrinsically disordered polypeptides derived from plant ureases

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.Fil: Grahl, Matheus V. Coste. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Lopes, Fernanda Cortez. Universidade Federal do Rio Grande do Sul; BrasilFil: Martinelli, Anne H. Souza. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R. S.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Coprophagous features in carnivorous Nepenthes plants: A task for ureases

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy

Soyuretox, an intrinsically disordered polypeptide derived from soybean (Glycine max) ubiquitous urease with potential use as a biopesticide

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.Fil: Kappaun, Karine. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Martinelli, Anne H. S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Zambelli, Barbara. Universidad de Bologna; ItaliaFil: Lopes, Fernanda C.. Universidade Federal do Rio Grande do Sul; BrasilFil: Ligabue-Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Moyetta, Natalia Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bonan, Carla D.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R. S.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Ciurli, Stefano. Universidad de Bologna; Itali

A New Role for Helicobacter pylori Urease: Contributions to Angiogenesis

Helicobacter pylori is a pathogen involved in gastric diseases such as ulcers and carcinomas. H. pylori’s urease is an important virulence factor produced in large amounts by this bacterium. In previous studies, we have shown that this protein is able to activate several cell types like neutrophils, monocytes, platelets, endothelial cells, and gastric epithelial cells. Angiogenesis is a physiological process implicated in growth, invasion and metastization of tumors. Here, we have analyzed the angiogenic potential of H. pylori urease (HPU) in gastric epithelial cells. No cytotoxicity was observed in AGS, Kato-III, and MKN28 gastric cell lines treated with 300 nM HPU, as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As we previously reported in neutrophils, treatment with 300 nM HPU also had an anti-apoptotic effect in gastric epithelial cells leading to a 2.2-fold increase in the levels of Bcl-XL after 6 h, and a decrease of 80% in the content of BAD, after 48 h, two mitochondrial proteins involved in regulation of apoptosis. Within 10 min of exposure, HPU is rapidly internalized by gastric epithelial cells. Treatment of the gastric cells with methyl-β-cyclodextrin abolished HPU internalization suggesting a cholesterol-dependent process. HPU induces the expression of pro-angiogenic factors and the decrease of expression of anti-angiogenic factors by AGS cells. The angiogenic activity of HPU was analyzed using in vitro and in vivo models. HPU induced formation of tube-like structures by human umbilical vascular endothelial cells in a 9 h experiment. In the chicken embryo chorioallantoic membrane model, HPU induced intense neo-vascularization after 3 days. In conclusion, our results indicate that besides allowing bacterial colonization of the gastric mucosa, H. pylori’s urease triggers processes that initiate pro-angiogenic responses in different cellular models. Thus, this bacterial urease, a major virulence factor, may also play a role in gastric carcinoma development

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053)\ud accumulate inside Callosobruchus maculatus larval midgut epithelium

It has been demonstrated that variant vicilins are the main resistance factor of cowpea seeds (Vigna unguiculata) against attack by the cowpea beetle Callosobruchus maculatus. There is evidence that the toxic properties of these storage proteins may be related to their interaction with glycoproteins and other microvillar membrane constituents along the digestive tract of the larvae. New findings have shown that following interaction with the microvilli, the vicilins are absorbed across the intestinal epithelium and thus reach the internal environment of the larvae. In the present paper we studied the insecticidal activity of the variant vicilins purified from a resistant cowpea variety (IT81D-1053). Bioassays showed that the seeds of this genotype affected larval growth, causing developmental retardation and 100% mortality. By feeding C. maculatus larvae on susceptible and IT81D-1053 derived vicilins (FITC labelled or unlabelled), followed by fluorescence and immunogold cytolocalization, we were able to demonstrate that both susceptible and variant forms are internalized in the midgut cells and migrate inside vesicular structures from the apex to the basal portion of the enterocytes. However, when larvae were fed with the labelled vicilins for 24 h and then returned to a control diet, the concentration of the variant form remained relatively high, suggesting that variant vicilins are not removed from the cells at the same rate as the non-variant vicilins. We suggest that the toxic effects of variant vicilins on midgut cells involve the binding of these proteins to the cell surface followed by internalization and interference with the normal physiology of the enterocytes, thereby affecting larval development in vivo

Most cited papers in Toxicon

Abstract Citation of a published work is one of the parameters considered in the analysis of relevance and importance of scientific contributions. In 2002, for the first time the Impact Factor of Toxicon has risen above 2.0, placing it at the 17th position among 76 journals in the 'toxicology' field. The aim of this article was to identify the most cited articles in Toxicon, that have contributed to the steady increase of its Impact Factor. The number of citations, complete reference and type of all documents appearing in Toxicon in the period 1963-2003 were retrieved from the ISI Web-of-Science homepage. The documents retrieved were sorted by the number of citations received. A 'citation index', defined as the number of citations divided by the number of years since publication, was calculated for each document. It was clearly seen that reviews in Toxicon received 4.4-fold more citations than articles. Unexpectedly, it was found that recent papers were proportionally more cited than old ones. A decrease in the proportion of papers dealing on 'snake p ' through out the period and the broadened range of subjects of the most cited papers recently published in Toxicon reflects an increased 'visibility' in other fields of toxinology. Research on plant toxins gained its own space in Toxicon with newer publications showing high citation indexes. It can be postulated that these facts helped to increase Toxicon's Impact Factor from 1.248 in 1999 to 2.003 in 2002. With the increased number of issues in Toxicon as well as publications of subject-dedicated volumes containing mostly reviews, the Impact Factor of Toxicon is expected to keep rising in the near future.

Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides

To meet the demands for food of the expanding world population, there is need of new ways for protecting plant crops against predators and pathogens while avoiding the use of environmentally aggressive chemicals. A milestone in this field was the introduction into crop plants of genes expressing Bacillus thuringiensis entomotoxic proteins. In spite of the success of this new technology, however, there are difficulties for acceptance of these ‘anti-natural’ products by the consumers and some concerns about its biosafety in mammals. An alternative could be exploring the plant’s own defense mechanisms, by manipulating the expression of their endogenous defense proteins, or introducing an insect control gene derived from another plant. This review deals with the biochemical features and mechanisms of actions of plant proteins supposedly involved in defense mechanisms against insects, including lectins, ribosome-inactivating proteins, enzymes inhibitors, arcelins, chitinases, ureases, and modified storage proteins. The potentialities of genetic engineering of plants with increased resistance to insect predation relying on the repertoire of genes found in plants are also discussed. Several different genes encoding plant entomotoxic proteins have been introduced into crop genomes and many of these insect resistant plants are now being tested in field conditions or awaiting commercialization

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [3H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-3H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.Fil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: De Martini, Diogo R.. Universidade Federal do Rio Grande do Sul; BrasilFil: Rubiolo, Edilberto Rene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Carlini, Célia Regina R S. 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; Argentin