Could the Urease of the Gut Bacterium Proteus mirabilis Play a Role in the Altered Gut-Brain Talk Associated with Parkinson's Disease?

Intestinal dysbiosis seems to play a role in neurodegenerative pathologies. Parkinson's disease (PD) patients have an altered gut microbiota. Moreover, mice treated orally with the gut microbe Proteus mirabilis developed Parkinson's-like symptoms. Here, the possible involvement of P. mirabilis urease (PMU) and its B subunit (PmUreß) in the pathogenesis of PD was assessed. Purified proteins were given to mice intraperitoneally (20 µg/animal/day) for one week. Behavioral tests were conducted, and brain homogenates of the treated animals were subjected to immunoassays. After treatment with PMU, the levels of TNF-α and IL-1ß were measured in Caco2 cells and cellular permeability was assayed in Hek 293. The proteins were incubated in vitro with α-synuclein and examined via transmission electron microscopy. Our results showed that PMU treatment induced depressive-like behavior in mice. No motor deficits were observed. The brain homogenates had an increased content of caspase-9, while the levels of α-synuclein and tyrosine hydroxylase decreased. PMU increased the pro-inflammatory cytokines and altered the cellular permeability in cultured cells. The urease, but not the PmUreß, altered the morphology of α-synuclein aggregates in vitro, forming fragmented aggregates. We concluded that PMU promotes pro-inflammatory effects in cultured cells. In vivo, PMU induces neuroinflammation and a depressive-like phenotype compatible with the first stages of PD development.

The neurotoxic mechanism of Jack Bean Urease in insects involves the interplay between octopaminergic and dopaminergic pathways.

In the last decades, the entomotoxicity of JBU and its derived peptides became an object of study, due mainly to the ubiquitous interaction of these compounds with different species of insects and their potential as natural insecticides. In this work, we investigated the neurotoxic effects of JBU in Nauphoeta cinerea cockroaches by dissecting pharmacologically the monoaminergic pathways involved. Selective pharmacological modulators for monoaminergic pathways in in vivo and ex vivo experimental models were employed. Thus, the analysis of N. cinerea neurolocomotory behavior demonstrated that JBU (1.5 and 3 µg/g) induces a significant decrease in the exploratory activity. In these assays, pretreatment of animals with phentolamine, SCH23390 or reserpine, interfered significantly with the response of JBU. Using in vivo abductor metathoracic preparations JBU (1.5 µg/g) induced progressive neuromuscular blockade, in 120 min recordings. In this set of experiments, the previous treatment of the animals with phentolamine, SCH23390 or reserpine, completely inhibited JBU-induced neuromuscular blockade. The recordings of spontaneous compound neural action potentials in N. cinerea legs showed that JBU, only in the smallest dose, significantly decreased the number of potentials in 60 min recordings. When the animals were pretreated with phentolamine, SCH23390, or reserpine, but not with mianserin, there was a significant prevention of the JBU-inhibitory responses on the action potentials firing. Meanwhile, the treatment of the animals with mianserin did not affect JBU's inhibitory activity. The data presented in this work strongly suggest that the neurotoxic response of JBU in N. cinerea involves a cross talking between OCTOPAMIN-ergic and DOPAMIN-ergic nerve systems, but not the SEROTONIN-ergic neurotransmission. Further molecular biology studies with expression of insect receptors associated with voltage clamp techniques will help to discriminate the selectivity of JBU over the monoaminergic transmission.

Helicobacter pylori Urease: Potential Contributions to Alzheimer's Disease.

Alzheimer's disease (AD) causes dementia and memory loss in the elderly. Deposits of beta-amyloid peptide and hyperphosphorylated tau protein are present in a brain with AD. A filtrate of Helicobacter pylori's culture was previously found to induce hyperphosphorylation of tau in vivo, suggesting that bacterial exotoxins could permeate the blood-brain barrier and directly induce tau's phosphorylation. H. pylori, which infects ~60% of the world population and causes gastritis and gastric cancer, produces a pro-inflammatory urease (HPU). Here, the neurotoxic potential of HPU was investigated in cultured cells and in rats. SH-SY5Y neuroblastoma cells exposed to HPU (50-300 nM) produced reactive oxygen species (ROS) and had an increased [Ca2+]i. HPU-treated BV-2 microglial cells produced ROS, cytokines IL-1ß and TNF-α, and showed reduced viability. Rats received daily i.p., HPU (5 µg) for 7 days. Hyperphosphorylation of tau at Ser199, Thr205 and Ser396 sites, with no alterations in total tau or GSK-3ß levels, and overexpression of Iba1, a marker of microglial activation, were seen in hippocampal homogenates. HPU was not detected in the brain homogenates. Behavioral tests were performed to assess cognitive impairments. Our findings support previous data suggesting an association between infection by H. pylori and tauopathies such as AD, possibly mediated by its urease.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system.

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Soluble Epoxide Hydrolase and Brain Cholesterol Metabolism.

The bifunctional enzyme soluble epoxide hydrolase (sEH) is found in all regions of the brain. It has two different catalytic activities, each assigned to one of its terminal domains: the C-terminal domain presents hydrolase activity, whereas the N-terminal domain exhibits phosphatase activity. The enzyme's C-terminal domain has been linked to cardiovascular protective and anti-inflammatory effects. Cholesterol-related disorders have been associated with sEH, which plays an important role in the metabolism of cholesterol precursors. The role of sEH's phosphatase activity has been so far poorly investigated in the context of the central nervous system physiology. Given that brain cholesterol disturbances play a role in the onset of Alzheimer's disease (AD) as well as of other neurodegenerative diseases, understanding the functions of this enzyme could provide pivotal information on the pathophysiology of these conditions. Moreover, the sEH phosphatase domain could represent an underexplored target for drug design and therapeutic strategies to improve symptoms related to neurodegenerative diseases. This review discusses the function of sEH in mammals and its protein structure and catalytic activities. Particular attention was given to the distribution and expression of sEH in the human brain, deepening into the enzyme's phosphatase activity and its participation in brain cholesterol synthesis. Finally, this review focused on the metabolism of cholesterol and its association with AD.

Ureases: Historical aspects, catalytic, and non-catalytic properties - A review.

Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme produced by plants, fungi, and bacteria that catalyzes the hydrolysis of urea into ammonia and carbamate. Urease is of historical importance in Biochemistry as it was the first enzyme ever to be crystallized (1926). Finding nickel in urease's active site (1975) was the first indication of a biological role for this metal. In this review, historical and structural features, kinetics aspects, activation of the metallocenter and inhibitors of the urea hydrolyzing activity of ureases are discussed. The review also deals with the non-enzymatic biological properties, whose discovery 40 years ago started a new chapter in the study of ureases. Well recognized as virulence factors due to the production of ammonia and alkalinization in diseases by urease-positive microorganisms, ureases have pro-inflammatory, endocytosis-inducing and neurotoxic activities that do not require ureolysis. Particularly relevant in plants, ureases exert insecticidal and fungitoxic effects. Data on the jack bean urease and on jaburetox, a recombinant urease-derived peptide, have indicated that interactions with cell membrane lipids may be the basis of the non-enzymatic biological properties of ureases. Altogether, with this review we wanted to invite the readers to take a second look at ureases, very versatile proteins that happen also to catalyze the breakdown of urea into ammonia and carbamate.

Jaburetox: update on a urease-derived peptide.

Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jbureII gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 µg it provoked death of Triatoma infestans bugs. JBTX (10-5-10-6 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.

Jaburetox: update on a urease-derived peptide

Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 μg it provoked death of Triatoma infestans bugs. JBTX (10−5-10−6 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.(AU)

Jaburetox: update on a urease-derived peptide

Abstract Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 g it provoked death of Triatoma infestans bugs. JBTX (105-106 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.

Jaburetox: update on a urease-derived peptide

Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 μg it provoked death of Triatoma infestans bugs. JBTX (10−5-10−6 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.(AU)

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.

Microcephaly and Zika virus: a clinical and epidemiological analysis of the current outbreak in Brazil / Microcefalia e vírus Zika: um olhar clínico e epidemiológico do surto em vigênciano Brasil

Abstract Objective: This study aimed to critically review the literature available regarding the Zika virus outbreak in Brazil and its possible association with microcephaly cases. Sources: Experts from Instituto do Cérebro do Rio Grande do Sul performed a critical (nonsystematic) literature review regarding different aspects of the Zika virus outbreak in Brazil, such as transmission, epidemiology, diagnostic criteria, and its possible association with the increase of microcephaly reports. The PubMed search using the key word “Zika virus” in February 2016 yielded 151 articles. The manuscripts were reviewed, as well as all publications/guidelines from the Brazilian Ministry of Health, World Health Organization and Centers for Disease Control and Prevention (CDC – United States). Summary of findings: Epidemiological data suggest a temporal association between the increased number of microcephaly notifications in Brazil and outbreak of Zika virus, primarily in the Brazil's Northeast. It has been previously documented that many different viruses might cause congenital acquired microcephaly. Still there is no consensus on the best curve to measure cephalic circumference, specifically in preterm neonates. Conflicting opinions regarding the diagnosis of microcephaly (below 2 or 3 standard deviations) that should be used for the notifications were also found in the literature. Conclusion: The development of diagnostic techniques that confirm a cause–effect association and studies regarding the physiopathology of the central nervous system impairment should be prioritized. It is also necessary to strictly define the criteria for the diagnosis of microcephaly to identify cases that should undergo an etiological investigation.

Resumo Objetivos: O objetivo deste estudo foi realizar uma revisão crítica da literatura sobre o surto de vírus Zika no Brasil e sua possível associação com casos de microcefalia. Fonte de dados: Especialistas em áreas afins do Instituto do Cérebro do Rio Grande do Sul realizaram uma revisão crítica (não sistemática) da literatura sobre o vírus Zika, suas formas de transmissão, a epidemia no Brasil, critérios diagnósticos e a possível associação com os casos crescentes de microcefalia. O uso da palavra chave “Zika virus” na base de dados do PubMed em fevereiro de 2016, retorna 151 publicações. Estes textos foram revisados assim como todas as publicações e recomendações do Ministério da Saúde, Organização Mundial da Saúde e Centro de Controle de Doenças (CDC – USA). Síntese dos dados: Os dados epidemiológicos sugerem uma relação temporal entre aumento da notificação de casos de microcefalia e o surto de vírus Zika, principalmente no Nordeste do Brasil. Agentes virais comprovadamente podem ser causadores de microcefalia congênita adquirida. Não existe um consenso sobre a melhor curva de perímetro cefálico a ser utilizada, principalmente nos prematuros. Assim como também existem divergências sobre a definição de microcefalia (abaixo de 2 ou 3 desvios padrões) a ser padronizada nas notificações. Conclusão: Deve-se priorizar o desenvolvimento de técnicas diagnósticas que confirmem uma relação causa–efeito e estudos sobre mecanismos da patogênese da infecção pelo Zika no sistema nervoso central. Também é necessário definir e universalizar os critérios diagnósticos para a identificação dos casos de microcefalia que devem ser submetidos à investigação etiológica.

Microcephaly and Zika virus: a clinical and epidemiological analysis of the current outbreak in Brazil.

OBJECTIVE: This study aimed to critically review the literature available regarding the Zika virus outbreak in Brazil and its possible association with microcephaly cases. SOURCES: Experts from Instituto do Cérebro do Rio Grande do Sul performed a critical (nonsystematic) literature review regarding different aspects of the Zika virus outbreak in Brazil, such as transmission, epidemiology, diagnostic criteria, and its possible association with the increase of microcephaly reports. The PubMed search using the key word "Zika virus" in February 2016 yielded 151 articles. The manuscripts were reviewed, as well as all publications/guidelines from the Brazilian Ministry of Health, World Health Organization and Centers for Disease Control and Prevention (CDC - United States). SUMMARY OF FINDINGS: Epidemiological data suggest a temporal association between the increased number of microcephaly notifications in Brazil and outbreak of Zika virus, primarily in the Brazil's Northeast. It has been previously documented that many different viruses might cause congenital acquired microcephaly. Still there is no consensus on the best curve to measure cephalic circumference, specifically in preterm neonates. Conflicting opinions regarding the diagnosis of microcephaly (below 2 or 3 standard deviations) that should be used for the notifications were also found in the literature. CONCLUSION: The development of diagnostic techniques that confirm a cause-effect association and studies regarding the physiopathology of the central nervous system impairment should be prioritized. It is also necessary to strictly define the criteria for the diagnosis of microcephaly to identify cases that should undergo an etiological investigation.

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

Role of lysine and acidic amino acid residues on the insecticidal activity of Jackbean urease.

Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine-borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus. The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.

3-to-1: unraveling structural transitions in ureases.

Ureases are nickel-dependent enzymes which catalyze the hydrolysis of urea to ammonia and carbamate. Despite the apparent wealth of data on ureases, many crucial aspects regarding these enzymes are still unknown, or constitute matter for ongoing debates. One of these is most certainly their structural organization: ureases from plants and fungi have a single unit, while bacterial and archaean ones have three-chained structures. However, the primitive state of these proteins--single- or three-chained--is yet unknown, despite many efforts in the field. Through phylogenetic inference using three different datasets and two different algorithms, we were able to observe chain number transitions displayed in a 3-to-1 fashion. Our results imply that the ancestral state for ureases is the three-chained organization, with single-chained ureases deriving from them. The two-chained variants are not evolutionary intermediates. A fusion process, different from those already studied, may explain this structural transition.

Evidence-based docking of the urease activation complex.

Ureases require accessory proteins for their activation and proper function. In Klebsiella aerogenes, UreD, UreF, UreG, and UreE are sequentially complexed to UreABC as required for its activation. Until now, only low-resolution structures are available for this activation complex. To circumvent such limitation, our work intends to provide an atomic-level model for the (UreABC-UreDFG)3 complex from K. aerogenes, by employing comparative modeling associated to sequential macromolecular dockings, validated through small-angle X-ray scattering profiles and comparison with results from cross-linking, mutagenesis, and pull-down experiments. Additionally, normal mode analyses of the obtained complex supported the characterization of the elevated flexibility of both UreD-UreF dimer and (UreABC-UreDFG)3 oligomer, explaining the previously observed diffuse binding of UreD to the apoenzyme. The model shown here is the first atomic-level depiction of this complex, a required step for the unraveling of the urease activation process. (1)Both authors share senior authorship. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:6.

Fungitoxic and insecticidal plant polypeptides.

According to the World Bank and FAO, the population grows worldwide and the poorest countries are expected to double their population within the next decades, reaching approximately 7.2 billion in 2015. Moreover, the food and financial crisis together with the global economic recession pushed the number of hungry and undernourished people in the world to unprecedented levels. The substitution of animal proteins by plant proteins in food and feed is a general trend because of the lower cost and better production efficiency. Pathogens and pests can reduce the crop yields up to 30%. In some places, the losses can reach 80% due to climate conditions, proliferation of insects, and fungal diseases. All together, the harvest and postharvest losses vary from 5% to 20% and depending on the commodity can be as high as 50%. Plants have a complex chemical armory for defense composed of low and high molecular mass compounds that can act over a variety of pests and pathogens, from micro-organisms to phytophagous insects or grazing animals. Among them, plant fungitoxic and insecticidal polypeptides represent promising alternatives to increase the supply of plant-derived proteins and tackle the hunger in a global scale.