Rodrigo Ligabue-Braun

Venomous mammals: a review.

The occurrence of venom in mammals has long been considered of minor importance, but recent fossil discoveries and advances in experimental techniques have cast new light into this subject. Mammalian venoms form a heterogeneous group having different compositions and modes of action and are present in three classes of mammals, Insectivora, Monotremata, and Chiroptera. A fourth order, Primates, is proposed to have venomous representatives. In this review we highlight recent advances in the field while summarizing biochemical characteristics of these secretions and their effects upon humans and other animals. Historical aspects of venom discovery and evolutionary hypothesis regarding their origin are also discussed.

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 β-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 β-hairpin motif (residues 61-74), JbtxΔ-β; 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-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-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 β-hairpin motif. Although the β-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.

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

Ureases (EC 3.5.1.5) are Ni2+‐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).

Antifungal properties of Canavalia ensiformis urease and derived peptides.

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.

Canavalia ensiformis urease, Jaburetox and derived peptides form ion channels in planar lipid bilayers.

Ureases catalyze the hydrolysis of urea into NH3 and CO2. 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 7-18pS and 32-79pS, 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.

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

Abstract 1 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. 1Both authors share senior authorship. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:6

Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses

Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.

Structural and transcriptional characterization of a novel member of the soybean urease gene family.

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s).

The Calcium Goes Meow: Effects of Ions and Glycosylation on Fel d 1, the Major Cat Allergen.

The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca2+ binding sites. Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy.