Jaburetox, a urease-derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea.

Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.

Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease.

BACKGROUND: Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent ß-hairpin motif consistent with either neurotoxicity or pore formation. METHODS: Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the ß-hairpin motif (residues 61-74), JbtxΔ-ß; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter. RESULTS: 1) JbtxΔ-ß disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the ß-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis. MAJOR CONCLUSIONS: The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the ß-hairpin motif. Although the ß-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. GENERAL SIGNIFICANCE: Jbtx represents a new type of insecticidal and membrane-active peptide.

Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas' disease vector.

Jaburetox, a recombinant peptide of ∼11kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas' disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae).

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.

Structural analysis of the interaction between Jaburetox, an intrinsically disordered protein, and membrane models.

Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.

Humoral and cellular immune responses induced by the urease-derived peptide Jaburetox in the model organism Rhodnius prolixus.

BACKGROUND: Although the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species. RESULTS: The findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide. CONCLUSIONS: In our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox's mechanism of action.

Interaction of jack bean (Canavalia ensiformis) urease and a derived peptide with lipid vesicles.

Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox "domain" into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU.

Pliable natural biocide: Jaburetox is an intrinsically disordered insecticidal and fungicidal polypeptide derived from jack bean urease.

Jaburetox is a polypeptide derived from jack bean (Canavalia ensiformis) urease and toxic to a broad spectrum of insects, phytopathogenic filamentous fungi and yeasts of medical importance. The elucidation of the structural basis for the mode of action of Jaburetox is the focus of this multifaceted study. Jaburetox in solution is a monomer of 11.0 kDa featuring a large hydrodynamic radius, suggestive of a disordered polypeptide. The intrinsically disordered nature of Jaburetox was theoretically predicted by a comprehensive bioinformatics analysis and experimentally confirmed by light scattering as well as by circular dichroism and NMR spectroscopy. NMR signal assignment provided backbone secondary chemical shifts that indicated that Jaburetox has a low propensity to assume a stable secondary structure. (15)N relaxation studies revealed significant backbone mobility, especially in the N-terminal portion of the polypeptide. The solution structure of Jaburetox shows the presence of an α-helical motif close to the N terminus, together with two turn-like structures situated in the central portion of the protein and close to the C terminus. Similar regions were predicted as potential protein-protein interaction sites using computational tools. The knowledge of the structural properties of Jaburetox in solution is a key step to correlate its structural and biological activities.

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.