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Célia R. Carlini - One of the best experts on this subject based on the ideXlab platform.
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Ureases as multifunctional toxic proteins: A review
Toxicon : official journal of the International Society on Toxinology, 2015Co-Authors: Célia R. Carlini, Rodrigo Ligabue-braunAbstract:Ureases are metalloenzymes that hydrolyze urea into ammonia and carbon dioxide. They were the first enzymes to be crystallized and, with them, the notion that enzymes are proteins became accepted. Novel toxic properties of Ureases that are independent of their enzyme activity have been discovered in the last three decades. Since our first description of the neurotoxic properties of canatoxin, an isoform of the jack bean Urease, which appeared in Toxicon in 1981, about one hundred articles have been published on "new" properties of plant and microbial Ureases. Here we review the present knowledge on the non-enzymatic properties of Ureases. Plant Ureases and microbial Ureases are fungitoxic to filamentous fungi and yeasts by a mechanism involving fungal membrane permeabilization. Plant and at least some bacterial Ureases have potent insecticidal effects. This entomotoxicity relies partly on an internal peptide released upon proteolysis of ingested Urease by insect digestive enzymes. The intact protein and its derived peptide(s) are neurotoxic to insects and affect a number of other physiological functions, such as diuresis, muscle contraction and immunity. In mammal models some Ureases are acutely neurotoxic upon injection, at least partially by enzyme-independent effects. For a long time bacterial Ureases have been recognized as important virulence factors of diseases by Urease-producing microorganisms. Ureases activate exocytosis in different mammalian cells recruiting eicosanoids and Ca(2+)-dependent pathways, even when their ureolytic activity is blocked by an irreversible inhibitor. Ureases are chemotactic factors recognized by neutrophils (and some bacteria), activating them and also platelets into a pro-inflammatory "status". Secretion-induction by Ureases may play a role in fungal and bacterial diseases in humans and other animals. The now recognized "moonlighting" properties of these proteins have renewed interest in Ureases for their biotechnological potential to improve plant defense against pests and as potential targets to ameliorate diseases due to pathogenic Urease-producing microorganisms.
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jack bean canavalia ensiformis Urease induces eicosanoid modulated hemocyte aggregation in the chagas disease vector rhodnius prolixus
Toxicon, 2014Co-Authors: Marina S Defferrari, Célia R. Carlini, Ian Orchard, R Da SilvaAbstract:Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean Urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to Ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time Urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein.
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canavalia ensiformis Urease jaburetox and derived peptides form ion channels in planar lipid bilayers
Archives of Biochemistry and Biophysics, 2014Co-Authors: Angela Regina Piovesan, Rodrigo Ligabuebraun, Anne H S Martinelli, Jeanlouis Schwartz, Célia R. CarliniAbstract:Abstract Ureases catalyze the hydrolysis of urea into NH 3 and CO 2 . They are synthesized by plants, fungi and bacteria but not by animals. Ureases display biological activities unrelated to their enzymatic activity, i.e., platelet and neutrophil activation, fungus inhibition and insecticidal effect. Urease from Canavalia ensiformis (jack bean) is toxic to several hemipteran and coleopteran insects. Jaburetox is an insecticidal fragment derived from jack bean Urease. Among other effects, Jaburetox has been shown to interact with lipid vesicles. In this work, the ion channel activity of C. ensiformis Urease, Jaburetox and three deletion mutants of Jaburetox (one lacking the N-terminal region, one lacking the C-terminal region and one missing the central β-hairpin) were tested on planar lipid bilayers. All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states whose conductance ranges were 7–18 pS and 32–79 pS, respectively. Urease and the N-terminal mutant of Jaburetox were more active at negative potentials, while the channels of the other peptides did not display voltage-dependence. This is the first direct demonstration of the capacity of C. ensiformis Urease and Jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be a crucial step of their diverse biological activities, including host defense.
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antifungal properties of canavalia ensiformis Urease and derived peptides
Peptides, 2012Co-Authors: Melissa Postal, Valdirene Moreira Gomes, Anne H S Martinelli, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Suzanna F F Ribeiro, Giancarlo Pasquali, Célia R. CarliniAbstract:Abstract Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO 2 . These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of Urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli . The antifungal activity of Ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis Urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H + and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to Ureases. Plant Ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10 −6 –10 −7 M.
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characterization of jbure iib isoform of canavalia ensiformis l dc Urease
Biochimica et Biophysica Acta, 2011Co-Authors: Fernanda Mulinari, Célia R. Carlini, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Fernanda Staniscuaski, Hugo Verli, Rodrigo R Fragoso, Evelyn Koeche Schroeder, Maria Fatima GrossidesaAbstract:Abstract Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis Urease isoform named JBURE-II, corresponding to a putative smaller Urease protein (78 kDa) when compared to other plant Ureases. Aiming to produce the recombinant protein, we obtained jbure - IIb , with different 3′ and 5′ ends, encoding a 90 kDa Urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure - II /- IIb is a functional gene. Comparative modeling indicates that JBURE-IIb Urease has an overall shape almost identical to C . ensiformis major Urease JBURE-I with all residues critical for Urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli . The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apoUrease are consistent with a protective role of Ureases in plants.
Robert A. Burne - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Urease Expression in Actinomyces naeslundii WVU45
Infection and Immunity, 2000Co-Authors: Evangelia Morou-bermudez, Robert A. BurneAbstract:The hydrolysis of urea by Ureases of oral bacteria in dental plaque can cause a considerable increase in plaque pH, which can inhibit the development of dental caries. There is also indirect evidence that urea metabolism may promote the formation of calculus and that ammonia release from urea could exacerbate periodontal diseases. Actinomyces naeslundii, an early colonizer of the oral cavity and a numerically significant plaque constituent, demonstrates comparatively low levels of Urease activity on isolation, so this organism has not been considered a major contributor to total oral Urease activity. In this study it was observed that Urease activity and Urease-specific mRNA levels in A. naeslundii WVU45 can increase up to 50-fold during growth under nitrogen-limiting conditions. Using primer extension analysis, a putative, proximal, nitrogen-regulated promoter of the A. naeslundii Urease gene cluster was identified. The functionality and nitrogen responsiveness of this promoter were confirmed using reporter gene fusions and 5' deletion analysis. The data indicated that regulation of Urease expression by nitrogen availability in A. naeslundii may require a positive transcriptional activator. Plaque bacteria may experience nitrogen limitation when carbohydrates are present in excess. Therefore, based on the results of this study and in contrast to previous beliefs, strains of A. naeslundii may have the potential to be significant contributors to total plaque ureolysis, particularly during periods when there is an increased risk for caries development.
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analysis of streptococcus salivarius Urease expression using continuous chemostat culture
Fems Microbiology Letters, 1996Co-Authors: Yiywan M Chen, Robert A. BurneAbstract:Alkali production from urea by bacterial Ureases in the oral cavity is thought to have a major impact on oral health and on the physiology and ecology of oral bacteria. Using continuous chemostat culture, Urease activity in Streptococcus salivarius 57.I was examined as a function of growth pH, carbohydrate availability and growth rate. A portion of the S. salivarius ureC gene was amplified by polymerase chain reactions (PCRs) using degenerate primers encoding highly conserved sequences from known Ureases. The nucleotide sequence of the PCR product was determined, and was used to compare the level of Urease gene expression under different growth conditions. The data indicated that Urease was highly expressed at low pH, and expression was also modulated by glucose availability and growth rate. Differential expression was controlled, at least in part, at the transcriptional level.
Cristian Follmer - One of the best experts on this subject based on the ideXlab platform.
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Cysteine based novel noncompetitive inhibitors of Urease(s)--distinctive inhibition susceptibility of microbial and plant Ureases.
Bioorganic & medicinal chemistry, 2006Co-Authors: Zareen Amtul, Cristian Follmer, Naheed Kausar, Richard Rozmahel, Atta-ur-rahman, Syed Arif Kazmi, Mohammed Saleh Shekhani, Jason L. Eriksen, Khalid Mohammed Khan, M. I. ChoudharyAbstract:Abstract Based on the catalysis mechanism of Urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial Ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant Urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit Urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak Urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver–Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum Ki value of 2 μM for N,N-dimethyl l -cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial Ureases, compared with the plant-originated Urease. Since microbial Urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential Urease inhibitors.
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separation of jack bean canavalia ensiformis Urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity
Plant Science, 2004Co-Authors: Cristian Follmer, German Enrique Wassermann, Célia R. CarliniAbstract:Abstract In this work we described the separation of two isoforms of Urease from jack bean seeds, the “classical” jack bean Urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean Urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)–Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the Urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains 1 eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these Ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both Ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean Urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators.
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Jackbean, soybean and Bacillus pasteurii Ureases
European journal of biochemistry, 2004Co-Authors: Cristian Follmer, Rafael Real-guerra, German Enrique Wasserman, Deiber Olivera-severo, Célia R. CarliniAbstract:In this work we compared two plant Ureases, jackbean Urease (JBU) and embryo-specific soybean Urease (SBU) and a bacterial (Bacillus pasteurii) Urease, for kinetic parameters and other biological properties described recently for Ureases that are independent of the ureolytic activity. The insecticidal effect of Ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii Urease (PBU), both plant Ureases presented potent insecticidal activity, with LD50 values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin – a Urease isoform from jackbean seeds that displays a toxic effect in mice (LD50 = 2 mg·kg−1) – no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg·kg−1). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC50 = 400.0 µg·mL−1, 22.2 µg·mL−1, 15.8 µg·mL−1 for BPU, SBU and JBU, respectively). This platelet activating property was also independent of Urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni+2 and Zn+2 ions. The Ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean Ureases and that these proteins may have a role in plant defense against insect predators.
Diogo Ribeiro Demartini - One of the best experts on this subject based on the ideXlab platform.
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antifungal properties of canavalia ensiformis Urease and derived peptides
Peptides, 2012Co-Authors: Melissa Postal, Valdirene Moreira Gomes, Anne H S Martinelli, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Suzanna F F Ribeiro, Giancarlo Pasquali, Célia R. CarliniAbstract:Abstract Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO 2 . These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of Urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli . The antifungal activity of Ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis Urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H + and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to Ureases. Plant Ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10 −6 –10 −7 M.
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characterization of jbure iib isoform of canavalia ensiformis l dc Urease
Biochimica et Biophysica Acta, 2011Co-Authors: Fernanda Mulinari, Célia R. Carlini, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Fernanda Staniscuaski, Hugo Verli, Rodrigo R Fragoso, Evelyn Koeche Schroeder, Maria Fatima GrossidesaAbstract:Abstract Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis Urease isoform named JBURE-II, corresponding to a putative smaller Urease protein (78 kDa) when compared to other plant Ureases. Aiming to produce the recombinant protein, we obtained jbure - IIb , with different 3′ and 5′ ends, encoding a 90 kDa Urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure - II /- IIb is a functional gene. Comparative modeling indicates that JBURE-IIb Urease has an overall shape almost identical to C . ensiformis major Urease JBURE-I with all residues critical for Urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli . The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apoUrease are consistent with a protective role of Ureases in plants.
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insecticidal effect of canavalia ensiformis major Urease on nymphs of the milkweed bug oncopeltus fasciatus and characterization of digestive peptidases
Insect Biochemistry and Molecular Biology, 2011Co-Authors: Marina S Defferrari, Diogo Ribeiro Demartini, Thiago Beltram Marcelino, Paulo Marcos Pinto, Célia R. CarliniAbstract:Jackbean (Canavalia ensiformis) Ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean Urease (JBU). O. fasciatus nymphs fed Urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within Urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the Urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean Urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.
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characterization of jbure iib isoform of canavalia ensiformis l dc Urease proteins and proteomics
Biochimica et Biophysica Acta, 2011Co-Authors: Fernanda Mulinari, Célia R. Carlini, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Fernanda Staniscuaski, Hugo Verli, Evelyn Koeche Schroeder, Rodrigo Da Rocha Fragoso, Maria Fatima GrossidesaAbstract:Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis Urease isoform named JBURE-II, corresponding to a putative smaller Urease protein (78kDa) when compared to other plant Ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3′ and 5′ ends, encoding a 90kDa Urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb Urease has an overall shape almost identical to C. ensiformis major Urease JBURE-I with all residues critical for Urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apoUrease are consistent with a protective role of Ureases in plants.
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global and targeted proteomics in developing jack bean canavalia ensiformis seedlings an investigation of Urease isoforms mobilization in early stages of development
Plant Molecular Biology, 2011Co-Authors: Célia R. Carlini, Diogo Ribeiro Demartini, Jay J ThelenAbstract:Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from Ureases in the midgut of susceptible insects. To characterize expression of Urease isoforms in jack bean seed, particularly the more abundant Urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti–JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS–PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC–MS/MS). Acquired MS2 data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted Urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of Urease isoforms during the early stages of plant development.
Anne H S Martinelli - One of the best experts on this subject based on the ideXlab platform.
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soybean ubiquitous Urease with purification facilitator an addition to the moonlighting studies toolbox
Process Biochemistry, 2017Co-Authors: Anne H S Martinelli, Rodrigo Ligabuebraun, Marina S Defferrari, Fernanda Cortez Lopes, Valquiria Broll, Karine Kappaun, Deise Michele Tichota, Leonardo L FrutteroAbstract:Abstract 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.
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canavalia ensiformis Urease jaburetox and derived peptides form ion channels in planar lipid bilayers
Archives of Biochemistry and Biophysics, 2014Co-Authors: Angela Regina Piovesan, Rodrigo Ligabuebraun, Anne H S Martinelli, Jeanlouis Schwartz, Célia R. CarliniAbstract:Abstract Ureases catalyze the hydrolysis of urea into NH 3 and CO 2 . They are synthesized by plants, fungi and bacteria but not by animals. Ureases display biological activities unrelated to their enzymatic activity, i.e., platelet and neutrophil activation, fungus inhibition and insecticidal effect. Urease from Canavalia ensiformis (jack bean) is toxic to several hemipteran and coleopteran insects. Jaburetox is an insecticidal fragment derived from jack bean Urease. Among other effects, Jaburetox has been shown to interact with lipid vesicles. In this work, the ion channel activity of C. ensiformis Urease, Jaburetox and three deletion mutants of Jaburetox (one lacking the N-terminal region, one lacking the C-terminal region and one missing the central β-hairpin) were tested on planar lipid bilayers. All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states whose conductance ranges were 7–18 pS and 32–79 pS, respectively. Urease and the N-terminal mutant of Jaburetox were more active at negative potentials, while the channels of the other peptides did not display voltage-dependence. This is the first direct demonstration of the capacity of C. ensiformis Urease and Jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be a crucial step of their diverse biological activities, including host defense.
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antifungal properties of canavalia ensiformis Urease and derived peptides
Peptides, 2012Co-Authors: Melissa Postal, Valdirene Moreira Gomes, Anne H S Martinelli, Arlete Beatriz Beckerritt, Rodrigo Ligabuebraun, Diogo Ribeiro Demartini, Suzanna F F Ribeiro, Giancarlo Pasquali, Célia R. CarliniAbstract:Abstract Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO 2 . These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of Urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli . The antifungal activity of Ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis Urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H + and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to Ureases. Plant Ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10 −6 –10 −7 M.
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Ubiquitous Urease affects soybean susceptibility to fungi
Plant Molecular Biology, 2012Co-Authors: Beatriz Wiebke-strohm, Anne H S Martinelli, Giancarlo Pasquali, Joseph C. Polacco, Márcia Margis-pinheiro, Marta Bencke, Lauro Bücker-neto, Arlete B. Becker-ritt, Ciliana Rechenmacher, Renata StolfAbstract:The soybean ubiquitous Urease (encoded by GmEu 4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant Ureases, notably in toxicity to other organisms. However, Urease enzymatic activity is not related to its toxicity. The role of GmEu 4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu 4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu 4, the transgenic plants exhibited GmEu 4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani , Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic Urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of Urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of Urease. It was not possible to overexpress active GmEu 4. For future work, overexpression of Urease fungitoxic peptides could be attempted as an alternative approach.
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antifungal activity of plant and bacterial Ureases
Toxicon, 2007Co-Authors: Arlete Beatriz Beckerritt, Anne H S Martinelli, Sydnei Mitidieri, Vanessa Feder, German Enrique Wassermann, Lucelia Santi, Marilene Henning Vainstein, Jose T A Oliveira, Lidia Mariana Fiuza, Giancarlo PasqualiAbstract:Ureases (EC 3.5.1.5) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Produced by plants, fungi and bacteria, but not by animals, Ureases share significant homology and similar mechanisms of catalysis, although differing in quaternary structures. While fungal and plant Ureases are homo-oligomeric proteins of 90 kDa subunits, bacterial Ureases are multimers of two (e.g. Helicobacter pylori) or three subunit complexes. It has been proposed that in plants these enzymes are involved in nitrogen bioavailability and in protection against pathogens. Previous studies by our group have shown that plant Ureases, but not a bacterial (Bacillus pasteurii) Urease, display insecticidal activity. Herein we demonstrate that (Glycine max) embryo-specific soybean Urease, jackbean (Canavalia ensiformis) major Urease and a recombinant H. pylori Urease impair growth of selected phytopathogenic fungi at sub-micromolar concentrations. This antifungal property of Ureases is not affected by treatment of the proteins with an irreversible inhibitor of the ureolytic activity. Scanning electron microscopy of Urease-treated fungi suggests plasmolysis and cell wall injuries. Altogether, our data indicate that Ureases probably contribute to the plant arsenal of defense compounds against predators and phytopathogens and that the Urease defense mechanism is independent of ammonia release from urea.
