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 10 kDa 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.     

Protein S-nitrosylation: Role for nitric oxide signaling in neuronal death

Background

One of the signaling mechanisms mediated by nitric oxide (NO) is through S-nitrosylation, the reversible redox-based modification of cysteine residues, on target proteins that regulate a myriad of physiological and pathophysiological processes. In particular, an increasing number of studies have identified important roles for S-nitrosylation in regulating cell death.

Scope of review

The present review focuses on different targets and functional consequences associated with nitric oxide and protein S-nitrosylation during neuronal cell death.

Major conclusions

S-Nitrosylation exhibits double-edged effects dependent on the levels, spatiotemporal distribution, and origins of NO in the brain: in general Snitrosylation resulting from the basal low level of NO in cells exerts anti-cell death effects, whereas S-nitrosylation elicited by induced NO upon stressed conditions is implicated in pro-cell death effects.

General Significance

Dysregulated protein S-nitrosylation is implicated in the pathogenesis of several diseases including degenerative diseases of the central nervous system (CNS). Elucidating specific targets of S-nitrosylation as well as their regulatory mechanisms may aid in the development of therapeutic intervention in a wide range of brain diseases.     

L925I Mutation in the Para-Type Sodium Channel Is Associated with Pyrethroid Resistance in Triatoma infestans from the Gran Chaco Region

Background

Chagas'' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil.The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species.

Methods and Findings

In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field.

Conclusions and Significance

The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas'' disease transmission.     

Inorganic phosphate uptake in unicellular eukaryotes

Background

Inorganic phosphate (P_i) is an essential nutrient for all organisms. The route of P_i utilization begins with P_i transport across the plasma membrane.

Scope of review

Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of P_i transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding P_i uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum.

Major conclusion

P_i uptake is the key step of P_i homeostasis and in the subsequent signaling event in eukaryotic microorganisms.

General significance

Biochemical and structural studies are important for clarifying mechanisms of P_i homeostasis, as well as P_i sensor and downstream pathways, and raise possibilities for future studies in this field.     

Electrochemical study of the intracellular transduction of vascular endothelial growth factor induced nitric oxide synthase activity using a multi-channel biocompatible microelectrode array

Background

Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array.

Methods

Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). N^G-nitro-l-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF.

Results

Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis.

Conclusion

Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase.

General significance

This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.     

Involvement of MAPK signaling pathway in the osteogenic gene expressions of Cervi Pantotrichum Cornu in MG-63 human osteoblast-like cells

Aims

The purposes of this study were to determine whether Cervi Pantotrichum Cornu (CPC) has osteogenic activities in human osteoblastic MG-63 cells and to investigate the underlying molecular mechanism.

Main methods

The effects of CPC on alkaline phosphatase activity, collagen synthesis, and calcium deposits were measured. The COL1A1, ALPL, BGLAP, and SPP1 expressions were measured by real-time PCR. Phosphorylated MAP kinases (ERK1/2, JNK1/2, p38, ELK1, and cJUN) were studied by western blot analysis. The involvement of MAPK pathway in osteogenic gene expressions was determined by using each selective MAPK inhibitor (PD98059, SP600125, and SB203580).

Key findings

CPC increased alkaline phosphatase activity, collagen synthesis, and calcium deposits. CPC activated ERK1/2, JNK1/2, p38, and ELK1 phosphorylation except cJUN. CPC increased the COL1A1, ALPL, BGLAP, and SPP1 gene expressions. The elevated COL1A1 and BGLAP expressions were inhibited by PD98059, SP600125 or SB203580. The elevated ALPL expression was blocked by SB203580. The elevated SPP1 expression was inhibited by SP600125 or SB203580. CPC increased COL1A1 and BGLAP expressions via ERK1/2, JNK1/2, and p38 MAPKs pathways and SPP1 expression via JNK1/2 and p38 pathways. p38 pathway is needed for ALPL expression.

Significance

These results imply that MAPK signaling pathway is an indispensable factor for bone matrix genes expression of CPC in MG-63 human osteoblast-like cells.     

Molecular basis of thermal stability in truncated (2/2) hemoglobins

Background

Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures.

Methods

We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position.

Results

The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible.

Conclusions

This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability.

General significance

These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.     

Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis

Background

The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity.

Methods

The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests.

Results

Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity.

Conclusions

An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II.

General significance

Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.     

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis

Background

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.

Methods

Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.

Results

We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.

Conclusions

In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.

General significance

Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.     

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer

Background

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods

Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Results

Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

Conclusions

As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

General significance

The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

The role of thioredoxin in the regulation of cellular processes by S-nitrosylation

Background

S-nitrosylation (or S-nitrosation) by Nitric Oxide (NO), i.e., the covalent attachment of a NO group to a cysteine thiol and formation of S-nitrosothiols (R-S-N=O or RSNO), has emerged as an important feature of NO biology and pathobiology. Many NO-related biological functions have been directly associated with the S-nitrosothiols and a considerable number of S-nitrosylated proteins have been identified which can positively or negatively regulate various cellular processes including signaling and metabolic pathways.

Scope of the review

Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by S-nitrosylation and Trx-mediated cellular homeostasis of S-nitrosothiols.

Major conclusions

Thioredoxin (Trx) system in mammalian cells utilizes thiol and selenol groups to maintain a reducing intracellular environment to combat oxidative/nitrosative stress. Reduced glutathione (GSH) and Trx system perform the major role in denitrosylation of S-nitrosylated proteins. However, under certain conditions, oxidized form of mammalian Trx can be S-nitrosylated and then it can trans-S-nitrosylate target proteins, such as caspase 3.

General significance

Investigations on the role of thioredoxin system in relation to biologically relevant RSNOs, their functions, and the mechanisms of S-denitrosylation facilitate the development of drugs and therapies. This article is part of a Special Issue entitled Regulation of Cellular Processes.