Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

An agonist of CXCR4 induces a rapid recovery from the neurotoxic effects of Vipera ammodytes and Vipera aspis venoms.

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA2 neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.

High-Throughput Venomics.

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

CC5 and CC8, Two Disintegrin Isoforms from Cerastes cerastes Snake Venom Decreased Inflammation Response In Vitro and In Vivo.

Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a promising strategy for disease prevention and treatment. In this study, we experimentally investigated the anti-inflammatory effect of CC5 and CC8, two disintegrin isoforms isolated from Cerastes cerastes snake venom, on LPS-stimulated macrophages, both on human THP-1 and mouse RAW264.7 cell adherence and their underlying mechanisms by measuring cytokine release levels and Western blot assay. Equally, both molecules were evaluated on a carrageenan-induced edema rat model. Our findings suggest that CC5 and CC8 were able to reduce adhesion of LPS-stimulated macrophages both on human THP-1 and mouse RAW264.7 cells to fibrinogen and vitronectin through the interaction with the αvß3 integrin receptor. Moreover, CC5 and CC8 reduced the levels of reactive oxygen species (ROS) mediated by the NF-κB, MAPK and AKT signaling pathways that lead to decreased production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 and increased secretion of IL-10 in LPS-stimulated THP-1 and RAW264.7 cells. Interestingly, both molecules potently exhibited an anti-inflammatory effect in vivo by reducing paw swelling in rats. In light of these results, we can propose the CC5 and CC8 disintegrins as interesting tools to design potential candidates against inflammatory-related diseases.

What's in a mass?

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake.

Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal ß-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.

Proof of concept: could snake venoms be a potential source of bioactive compounds for control of mould growth and mycotoxin production.

The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.

Characterization of a new member of kunitz-type protein family from the venom of Persian false-horned viper, Pseudocerastes persicus.

A new member of kunitz-type protein family, PPTI (PseudocerastesPersicusTrypsin Inhibitor), was isolated from the venom of Persian false horned viper Pseudocerastes persicus and characterized. Mass spectrometry and amino acid sequencing revealed that PPTI is a 68 amino acid protein with molecular weight of about 7.6 kDa. The first amino acid residue of PPTI is N-terminally blocked via a post translational modification to pyroglutamyl. Sequence comparison against UniProtKB shows a high sequence similarity of PPTI with kunitz-type proteins, especially serine protease inhibitors and dendrotoxins (DTXs). The number of cysteines and disulfide bonding pattern of PPTI are the same as kunitz-type proteins. Based on sequence derive information, anti-protease activity of PPTI against trypsin was experimentally examined. The constructed homology models of PPTI confirmed the ability of PPTI to fold similarly to kunitz domain. The presence of characteristic basic-hydrophobic functional dyad of DTXs in PPTI supports its inhibitory potential against potassium channels. In summary, this study hypothesized the dual functionality of PPTI according to its inhibitory effect on trypsin and its potential ability in blocking potassium channel.

Macrovipecetin, a C-type lectin from Macrovipera lebetina venom, inhibits proliferation migration and invasion of SK-MEL-28 human melanoma cells and enhances their sensitivity to cisplatin.

BACKGROUND: The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. METHODS: Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. RESULTS: Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced the expression of TRADD, p53, Bax, Bim and Bad and down-regulated the Bcl-2 expression and ROS levels in SK-MEL-28 cells. Interestingly, these treatments impaired SK-MEL-28 cell adhesion, migration and invasion through modulating the function and expression of αvß3 integrin along with regulating E-cadherin, vimentin, ß-catenin, c-Src and RhoA expression. In silico study suggested that only the α chain of macrovipecetin interacts with a region overlapping the RGD motif binding site on this integrin. CONCLUSIONS: We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells. GENERAL SIGNIFICANCE: The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.

Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

Inhibitory effects of ascorbic acid toward snake venom metalloproteinase (SVMP) from Indian Echis carinatus venom: Insights from molecular modeling and binding studies.

Classical antivenom therapy is unable to shield complications of viper bite and has limitations such as anaphylaxis and serum sickness. Snake venom metalloproteinases are responsible for local tissue damage and hemorrhage at the bitten site in viper envenomation, and this has led to a persistent search for metalloproteinase inhibitors. Here, we report the inhibitory effects of ascorbic acid against metalloproteinase from Echis carinatus venom both in-silico and in-vitro. Ascorbic acid effectively inhibited the proteolytic activity of E. carinatus venom in a dose-dependent manner. Interaction studies of ascorbic acid with purified ecarin using isothermal titration calorimetry showed favorable binding energy and energetics. The molecular docking of ascorbic acid with ecarin revealed important interactions with residues at the active site pocket of ecarin. It was observed that the ligand behaves as a chelating inhibitor. Thus, the backbone structural scaffold of ascorbic acid can find potential use as building blocks in designing drug-like molecules for viper bite management.

Toxin bioportides: exploring toxin biological activity and multifunctionality.

Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides-a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2.

Human Group IIA phospholipase A2 (hGIIA) promotes inflammation in immune-mediated pathologies by regulating the arachidonic acid pathway through both catalysis-dependent and -independent mechanisms. The hGIIA crystal structure, both alone and inhibitor-bound, together with structures of closely related snake-venom-derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis-independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A2 of Daboia russelli pulchella (svPLA2 ). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827-842. © 2016 Wiley Periodicals, Inc.

Protein toxins of the Echis coloratus viper venom directly activate TRPV1.

BACKGROUND: Peptide and protein toxins are essential tools to dissect and probe the biology of their target receptors. Venoms target vital physiological processes to evoke pain. Snake venoms contain various factors with the ability to evoke, enhance and sustain pain sensation. While a number of venom-derived toxins were shown to directly target TRPV1 channels expressed on somatosensory nerve terminals to evoke pain response, such toxins were yet to be identified in snake venoms. METHODS: We screened Echis coloratus saw-scaled viper venom's protein fractions isolated by reversed phase HPLC for their ability to activate TRPV1 channels. To this end, we employed heterologous systems to analyze TRPV1 and NGF pathways by imaging and electrophysiology, combined with molecular biology, biochemical, and pharmacological tools. RESULTS: We identified TRPV1 activating proteins in the venom of Echis coloratus that produce a channel-dependent increase in intracellular calcium and outwardly rectifying currents in neurons and heterologous systems. Interestingly, channel activation was not mediated by any of its known toxin binding sites. Moreover, although NGF neurotropic activity was detected in this venom, TRPV1 activation was independent of NGF receptors. CONCLUSIONS: Echis coloratus venom contains proteins with the ability to directly activate TRPV1. This activity is independent of the NGF pathway and is not mediated by known TRPV1 toxins' binding sites. GENERAL SIGNIFICANCE: Our results could facilitate the discovery of new toxins targeting TRPV1 to enhance current understanding of this receptor activation mechanism. Furthermore, the findings of this study provide insight into the mechanism through which snakes' venom elicit pain.

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.

Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.

Alterations of the immunosuppressive IL4I1 enzyme activity induced by naturally occurring SNP/mutations.

The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms.

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.