Molecular evidence for the evolution of ichnoviruses from ascoviruses by symbiogenesis

Background  

Female endoparasitic ichneumonid wasps inject virus-like particles into their caterpillar hosts to suppress immunity. These particles are classified as ichnovirus virions and resemble ascovirus virions, which are also transmitted by parasitic wasps and attack caterpillars. Ascoviruses replicate DNA and produce virions. Polydnavirus DNA consists of wasp DNA replicated by the wasp from its genome, which also directs particle synthesis. Structural similarities between ascovirus and ichnovirus particles and the biology of their transmission suggest that ichnoviruses evolved from ascoviruses, although molecular evidence for this hypothesis is lacking.     

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.     

Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum)

Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.Many species of aphids are important sucking-insect pests that feed on plant vascular fluids. Their feeding mechanism makes these insects excellent vectors for many plant pathogens, especially viruses, yet less amenable to standard, nonsystemic chemical control by insecticides. Minor effects on the survival and fecundity of aphids reared on Bacillus thuringiensis (Bt) crops have been noted in some studies but not in others (1, 3, 6). However, the sensitivity of aphids to Bt toxins, or the lack thereof, has not been previously tested through artificial-diet bioassays with exhaustive buffer-based controls.Bt δ-endotoxins Cyt1A, Cry4A/Cry4B, and Cry11, obtained from three recombinant strains of B. thuringiensis subsp. israelensis, as well as Cry1Ab and Cry3A, obtained from recombinant Escherichia coli, were purified by ultracentrifugation in a discontinuous sucrose gradient as described previously (9). Cry proteins were solubilized in solubilization buffer (50 mM Na₂CO₃, 100 mM NaCl, pH 10) with dithiothreitol (10 mM) added before use. Cyt1A was first solubilized on 10 mM Na₂CO₃ (pH 11) buffer and then neutralized at pH 7.5 to 8 with 10 μl HCl (1 N). Both solubilized and trypsin-digested samples (1:30 over toxin weight) were used at different concentrations (32, 125, and 500 μg/ml; trypsin-activated toxin concentrations were calculated on the basis of the preactivation concentrations of the protoxins) to supplement the AP3 aphid synthetic diet (7) used to feed Acyrthosiphon pisum (LL01 green clone). Ampicillin (100 μg/ml), an ineffective antibiotic for A. pisum or its obligate symbiont Buchnera, was added to the medium to avoid bacterial growth. For each concentration, 30 nymphs (10 nymphs/box and three repetitions) were bioassayed at 20°C and under a 16:8 (light-dark) photoperiod. Survival time was calculated from aphid deposition on the test diet (day 0). Mortality was surveyed daily, and body weights of survivors were noted at day 7. ST₅₀ (median survival time after challenge) was calculated by using an actuarial survival analysis (Statview) with censoring values of survivors at the end of the experiments. The approximate concentrations resulting in a 50% decrease in mean body weight (IC₅₀) and killing of 50% of the insects tested (LC₅₀) were calculated at the end of the experiments from the growth reduction and mortality data, respectively, derived with the three doses by using Statview and the censoring values of survivors.All of the Cry δ-endotoxins tested were lethal to A. pisum and retarded the growth of survivors (Fig. ​(Fig.11 and ​and2).2). Mortalities ranged from only 25% (Cry1Ab) to 100% (Cry4 and Cry11) after 3 to 6 days of exposure to 500 μg/ml of solubilized protein (Fig. ​(Fig.1).1). When significant mortalities were achieved (Cry3A, Cry4, and Cry11), trypsin activation enhanced toxicity. Activation of Cry4 at the intermediate concentration tested (125 μg/ml) resulted in a twofold increase in mortality (Fig. ​(Fig.1D).1D). ST₅₀s were calculated for both solubilized protoxins and activated Cry3A, Cry4, and Cry11. The ST₅₀s (at 500 μg/ml) ranged from 1.8 ± 0.14 days for solubilized Cry4 and Cry11 to 3.7 ± 1.2 days for trypsin-activated Cry3A (Table ​(Table1).1). Control aphids fed buffer all survived for >8 days. The LC₅₀ of Cry1Ab was not calculated, since mortality associated with Cry1Ab reached a plateau at 500 μg/ml. The LC₅₀ of Cry4 was estimated to be 70 to 100 μg/ml (data not shown).Open in a separate windowFIG. 1.Mortality assays over the nymphal life stage of the pea aphid, A. pisum, upon ingestion of artificial diets containing purified Bt toxins after either solubilization (open symbols) or solubilization and trypsin activation (solid symbols). The toxins used were Cry1Ab (circles), Cry3A (squares), a mixture of Cry4A and Cry4B (diamonds), and Cry11A (triangles). The soluble-toxin doses used were low at 32 μg/ml (blue), intermediate at 125 μg/ml (violet), and high at 500 μg/ml (red). Assays were carried out with 30 initial neonate insects in three batches of 10 individuals.Open in a separate windowFIG. 2.Growth inhibition assays with purified Bt toxins Cry3A, Cry4, and Cry 11 (A) and Cry1Ab and Cyt1A (B) on the pea aphid, A. pisum. Toxins were added to the diet either after solubilization (open symbols) or after solubilization and trypsin activation (solid symbols). Error bars show the standard errors (SE) of individual weights at day 7 of experiments, standardized by the control group mean weight (toxin dose, 0; initial number, 30). Color coding of toxins: Cry3A, red squares; Cry4A and Cry4B mixture, violet diamonds; Cry11A, blue triangles; Cry1Ab, green circles; Cyt1A, yellow squares. In the experiment with Cry1Ab (B), the toxin was purified by high-performance liquid chromatography and activated toxin was provided as a salt-free lyophilisate by W. Moar (Auburn University, Auburn, AL).

TABLE 1.

ST₅₀s of pea aphids feeding on solubilized Cry toxins and solubilized Cry toxins activated with trypsin

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113�C119

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.Approximately two million deaths in the world per year are caused by Plasmodium falciparum, the parasite responsible for tropical malaria (1, 2). In the last years, increasing interest has been developing for the peculiar glutathione S-transferase (PfGST)³ expressed by this parasite. Expressed in almost all living organisms, GSTs represent a large superfamily of multifunctional detoxifying enzymes that are able to conjugate GSH to a lot of toxic electrophilic compounds, thus facilitating their excretion. Many other protection roles of GSTs have been described, including the enzymatic reduction of organic peroxides (3–5), the inactivation of the proapoptotic JNK through a GST·JNK complex (6), and the protection of the cell from excess nitric oxide (7). The mammalian cytosolic GSTs are dimeric proteins grouped into eight species-independent classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta on the basis of sequence similarity, immunological reactivity, and substrate specificity (3, 8–11). PfGST is one of the most abundant proteins expressed by P. falciparum (from 1 to 10%, i.e. from 0.1 to 1 mm) (12), and different from what occurs in many organisms, it is the sole GST isoenzyme expressed by this parasite. Despite its structural similarity to the Mu class GST, this specific isoenzyme cannot be assigned to any known GST class (13). The interest in this enzyme is due to its particular protective role in the parasite. In fact, in addition to the usual GST activity that promotes the conjugation of GSH to electrophilic centers of toxic compounds, this protein efficiently binds hemin, and thus it could protect the parasite (that resides in the erythrocytes) from the parasitotoxic effect of this heme by-product (14). Specific compounds that selectively inhibit its catalytic activity or hemin binding could be promising candidates as antimalarial drugs. In this context, the discovery of structural or mechanistic properties of this enzyme that are not found in other GSTs may be important for designing selective inhibitors that are toxic to the parasite but harmless for the host cells. Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13, 16, 17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13, 18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix α-4 and helix α-5 (residues 113–119; see also Fig. 1) that could be involved in the dimer-dimer interaction. Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113–119 of one homodimer, which occupies an H-site of the other homodimer (13, 18). Upon binding of S-hexylglutathione, loop 113–119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix α-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17, 18). The changed course of residues 113–119 in the liganded enzyme prevents the interlocking of the dimers.Open in a separate windowFIGURE 1.A, structural changes of loop 113–119 occurring in the dimer (light blue model and yellow loop; Protein Data Bank code 2AAW) to tetramer (blue model and orange loop; Protein Data Bank code 1OKT) transition. Red spheres indicate the amino acids replaced in this study to obtain mutants A, B, and C. B, model of hemin·PfGST complex obtained by docking simulation using the crystal structure for Protein Data Bank code 1Q4J (15). Hemin is shown in red, loop 113–119 is in orange, and GSH is shown as yellow sticks.In this paper, by means of site-directed mutagenesis, fluorescence anisotropy, kinetic studies, and size exclusion chromatography, we check the influence of selected mutations of this atypic loop in the tetramerization process and the possible involvement of this protein segment in the cooperative phenomenon that characterizes hemin binding. In addition we describe that the tetramerization process is inhibited not only by GSH but even by GSSG. This finding suggests that hemin binding of PfGST is independent of the redox status of the cell. Finally, we demonstrate that the presence of GSH (or GSSG) in the active site is not essential for hemin binding, but this interaction only requires an active dimeric conformation.     

Symbiotic Virus at the Evolutionary Intersection of Three Types of Large DNA Viruses; Iridoviruses,Ascoviruses, and Ichnoviruses

Background

The ascovirus, DpAV4a (family Ascoviridae), is a symbiotic virus that markedly increases the fitness of its vector, the parasitic ichneumonid wasp, Diadromus puchellus, by increasing survival of wasp eggs and larvae in their lepidopteran host, Acrolepiopsis assectella. Previous phylogenetic studies have indicated that DpAV4a is related to the pathogenic ascoviruses, such as the Spodoptera frugiperda ascovirus 1a (SfAV1a) and the lepidopteran iridovirus (family Iridoviridae), Chilo iridescent virus (CIV), and is also likely related to the ancestral source of certain ichnoviruses (family Polydnaviridae).

Methodology/Principal Findings

To clarify the evolutionary relationships of these large double-stranded DNA viruses, we sequenced the genome of DpAV4a and undertook phylogenetic analyses of the above viruses and others, including iridoviruses pathogenic to vertebrates. The DpAV4a genome consisted of 119,343 bp and contained at least 119 open reading frames (ORFs), the analysis of which confirmed the relatedness of this virus to iridoviruses and other ascoviruses.

Conclusions

Analyses of core DpAV4a genes confirmed that ascoviruses and iridoviruses are evolutionary related. Nevertheless, our results suggested that the symbiotic DpAV4a had a separate origin in the iridoviruses from the pathogenic ascoviruses, and that these two types shared parallel evolutionary paths, which converged with respect to virion structure (icosahedral to bacilliform), genome configuration (linear to circular), and cytopathology (plasmalemma blebbing to virion-containing vesicles). Our analyses also revealed that DpAV4a shared more core genes with CIV than with other ascoviruses and iridoviruses, providing additional evidence that DpAV4a represents a separate lineage. Given the differences in the biology of the various iridoviruses and ascoviruses studied, these results provide an interesting model for how viruses of different families evolved from one another.     

A computational platform for MALDI-TOF mass spectrometry data: Application to serum and plasma samples

BackgroundMass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data.ResultsMASCAP integrates denoising and feature extraction algorithms, which have already shown to provide consistent peaks across mass spectra; furthermore, it relies on statistical analysis and graphical tools to compare the results between groups. The effectiveness in mass spectrum processing is demonstrated using MALDI-TOF data, as well as SELDI-TOF data. The usefulness in detecting potential protein biomarkers is shown comparing MALDI-TOF mass spectra collected from serum and plasma samples belonging to the same clinical population.ConclusionsThe analysis approach implemented in MASCAP may simplify biomarker detection, by assisting the recognition of proteomic expression signatures of the disease. A MATLAB implementation of the software and the data used for its validation are available at http://www.unich.it/proteomica/bioinf.     

Nuclear shield: a multi-enzyme task-force for nucleus protection

Background

In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions.

Methodology/Principal Findings

Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization.

Conclusions/Significance

The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes.     

The extended catalysis of glutathione transferase

Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ? [E]. As glutathione transferase lowers the pK_a of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed.     

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.