Receptor variability-driven evolution of snake toxins

Three-finger toxins(TFTs) are well-recognized nonenzymatic venom proteins found in snakes. However,although TFTs exhibit accelerated evolution, the drivers of this evolution remain poorly understood.The structural complexes between long-chainα-neurotoxins, a subfamily of TFTs, and their nicotinic acetylcholine receptor targets have been determined in previous research, providing an opportunity to address such questions. In the current study, we observed several previously identified positively selected sites(PSSs) and the highly variable C-terminal loop of these toxins at the toxin/receptor interface. Of interest, analysis of the molecular adaptation of the toxin-recognition regions in the corresponding receptors provided no statistical evidence for positive selection. However, these regions accumulated abundant amino acid variations in the receptors from the prey of snakes, suggesting that accelerated substitution of TFTs could be a consequence of adaptation to these variations. To the best of our knowledge, this atypical evolution, initially discovered in scorpions, is reported in snake toxins for the first time and may be applicable for the evolution of toxins from other venomous animals.     

Molecular evolution of snake venom toxins

Summary Phylogenetic trees were constructed for 62 venom toxins of snakes ofProteroglyphae suborder using matrix method. The resulting tree fromMinimum Spanning Tree-Cluster Analysis technique had the lowest percent deviation (8.55). The taxonomic relationship of these toxins agrees very well with zoological opinions. However, the appearance of the tree did not directly provide a plausible evolutionary model for the toxins. A model was derived from nodal ancestral sequence calculations, comparisons between intra-and inter-generical rates of amino acid change, and generally held ideas about protein evolution. According to the model, short neurotoxin is the ancient form of snake venom toxins. The courses of evolution leading to the present intraspecific homologous toxins are explained by gene duplication and allelomorphism.     

Molecular interactions between p-coumaric acid and snake venom toxins

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.     

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters.     

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.     

Peptide toxins that selectively target insect NaV and CaV channels

Numerous metazoans express venoms for the purpose of defense, competitor deterrence, or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology, and ion selectivity of insect voltage-gated sodium (Na_V) and calcium (Ca_V) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding. This review focuses on insect-selective peptide toxins and their utility for the study of insect Na_V and Ca_V channels. The high affinity and selectivity of some of these neurotoxins means that they have the potential to become the defining pharmacology for specific subtypes of insect ion channels. In addition, it might be possible to exploit the phyletic specificity of these toxins as the basis for rational development of novel classes of ion channel insecticides.     

Actions of snake neurotoxins on an insect nicotinic cholinergic synapse

Here we examine the actions of six snake neurotoxins (α-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneurone 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are senstive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes.     

Chlorotoxin and related peptides: Short insect toxins from scorpion venom

Scorpion venom is a complex multicomponent mixture of biologically active substances, some of which possess very interesting properties and are used in quite unexpected areas. The family of chlorotoxin (CTX)-like peptides is a good example. These toxins exhibit insecticidal activity, however, the molecular mechanism of their action on the insect organism has not been established yet. Nevertheless, CTX-like peptides attracted considerable research effort due to their ability to interact specifically with cells of malignant brain tumors—gliomas. In the future these compounds may significantly simplify treatment of tumor diseases. The review summarizes the results accumulated over a forty years period of CTX-like peptides study. Aspects of their natural functions are considered, as well as the application area associated with gliomas.     

Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution

Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.     

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.     

The progress in insect cross‐resistance among Bacillus thuringiensis toxins

Bt crop pyramids produce two or more Bt proteins active to broaden the spectrum of action and to delay the development of resistance in exposed insect populations. The cross‐resistance between Bt toxins is a vital restriction factor for Bt crop pyramids, which may reduce the effect of pyramid strategy. In this review, the status of the cross‐resistance among more than 20 Bt toxins that are most commonly used against 13 insect pests was analyzed. The potential mechanisms of cross‐resistance are discussed. The corresponding measures, including pyramid RNA interference and Bt toxin, “high dose/refuge,” and so on are advised to be taken for adopting the pyramided strategy to delay the Bt evolution of resistance and control the target pest insect.     

How Bacillus thuringiensis has evolved specific toxins to colonize the insect world

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation. Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.     

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body.     

All domains of Cry1A toxins insert into insect brush border membranes

A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only alpha-helix 5 of domain I along with adjacent helices alpha-4 or alpha-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of alpha-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.     

乌梢蛇血清对白眉蝮等3种蛇毒解毒作用初探

目的 探讨乌梢蛇血清对孟加拉眼镜蛇、白眉蝮、莽山烙铁头3种毒蛇的解毒作用。方法 给18～22g昆明小鼠分别注射盂加拉眼镜蛇毒、白眉蝮蛇毒及莽山烙铁头蛇毒后,随机分组注射不同浓度的乌梢蛇血清,并观察对3种蛇毒的解毒效果。结果 发现（1）乌梢蛇血清对盂加拉眼镜蛇毒有解毒作用．且注射蛇毒与4ml／kg的血清混合液的解毒作用最好,但随着间隔时间的延长,解毒作用逐渐减弱;（2）乌梢蛇血清对白眉蝮蛇毒有解毒作用,且注射量为1ml／kg和2ml／kg时出现了拮抗作用;（3）乌梢蛇血清对莽山烙铁头蛇毒有解毒作用,但莽山烙铁头蛇毒对血清注射量和间隔时间不敏感,在血清注射量为2ml／kg时和注射蛇毒10min后,再注射4ml／kg的血清时出现了拮抗作用。（4）只给小鼠注射不同量的乌梢蛇血清,发现乌梢蛇血清对小鼠无明显的毒副作用。结论 乌梢蛇血清对3种蛇毒均有解毒作用。     

Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.