RESUMEN

Snakebite envenomations result in acute and chronic physical and psychological health effects on their victims, leading to a substantial socio-economic burden in tropical and subtropical countries. Local necrosis is one of the serious effects caused by envenomation, primarily induced by snake venoms from the Viperidae family through the direct action of components collectively denominated as myotoxins, including the phopholipase A2-like (PLA2-like) toxins. Considering the limitations of antivenoms in preventing the rapid development of local tissue damage caused by envenomation, the use of small molecule therapeutics has been suggested as potential first-aid treatments or as adjuvants to antivenom therapy. In this review, we provide an overview of the structural interactions of molecules exhibiting inhibitory activity toward PLA2-like toxins. Additionally, we discuss the implications for the myotoxic mechanism of PLA2-like toxins and the molecules involved in their activation, highlighting key differences between activators and inhibitors. Finally, we integrate all these results to propose a classification of inhibitors into three different classes and five sub-classes. Taking into account the structural and affinity information, we compare the different inhibitors/ligands to gain a deeper understanding of the structural basis for the effective inhibition of PLA2-like toxins. By offering these insights, we aim to contribute to the search for new and efficient inhibitor molecules to complement and improve current therapy by conventional antivenoms.

RESUMEN

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.

Asunto(s)

Venenos de Crotálidos , Crotoxina , Serpientes Venenosas , Animales , Crotoxina/química , Fosfolipasas A2/química , Crotalus/metabolismo , Venenos de Crotálidos/química , Isoformas de Proteínas/metabolismo

RESUMEN

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Asunto(s)

ADN Glicosilasas , alfa Carioferinas , Animales , Ratones , Humanos , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Señales de Localización Nuclear/genética , ADN Glicosilasas/metabolismo

RESUMEN

Phospholipases A2 (PLA2s) are main components of snake venoms. Several snake species possess endogenous PLA2 inhibitors in their circulating blood, which are generally known as sbPLIs (an acronym for snake blood phospholipase A2inhibitors). The sbPLIs are categorized in three classes (alpha, beta or gamma) depending on the existence of distinguishing protein domains in their structure. The Crotalus durrissus terrificus venom has a highly neurotoxic PLA2 - crotoxin (CTX) - in its composition and the self-protection of the snake is mainly ensured by a sbγPLI named CNF (standing for Crotalusneutralizing factor). In an attempt to find smaller molecules able to inhibit the catalytic activity of CTX, in the present study we used linear peptide arrays to identify CNF segments possibly involved in the interaction with the toxin. Five reacting segments were identified as possible interacting regions. The target peptides were synthesized and located in the in silico CNF structure. Although all of them are exposed to the solvent, high concentrations were needed to inhibit the PLA2 activity of the whole venom or CTX. Limitations of the methodology employed and particular characteristics of CTX inhibition by CNF are discussed.

RESUMEN

Novel male contraceptives will promote gender equality in sharing contraceptive responsibility. The sperm-associated protein epididymal protease inhibitor (EPPIN) is a promising target for non-hormonal male contraception. EPPIN interacts with the semen coagulum protein semenogelin-1 (SEMG1) on the sperm surface, leading to transient inhibition of sperm motility after ejaculation. Small organic molecules targeting EPPIN's SEMG1-binding are under development as male contraceptives. Here, we combined computational approaches to uncover key aspects underlying EPPIN binding to SEMG1 and small organic ligands. We generated a human EPPIN model showing a typical arrangement of the WFDC (Whey-acid four disulfide core)-type and Kunitz-type domains, connected by a hinge region. Determining the EPPIN model's intrinsic motion by molecular dynamics simulations and normal mode analysis revealed a conformation, presenting a binding pocket that accommodates SEMG1Glu229-Gln247, EP055, and EP012. EPPIN's residues Phe63 and Lys68 (WFDC domain), Asp71 (hinge region), and Asn113, Asn114, and Asn115 (Kunitz domain) were identified as hot spots for SEMG1, EP055, and EP012 binding. Moreover, hydrophobic and hydrophilic residues in the WFDC and Kunitz domains allow plasma membrane anchoring, orienting the EPPIN binding pocket to the solvent. Targeting EPPIN's essential residues for its biomolecular interactions may improve the rational design of EPPIN ligands as spermiostatic compounds.

Asunto(s)

Anticonceptivos Masculinos , Humanos , Masculino , Anticonceptivos Masculinos/farmacología , Ligandos , Semen , Motilidad Espermática , Anticonceptivos

RESUMEN

Varespladib (LY315920) is a potent inhibitor of human group IIA phospholipase A2 (PLA2) originally developed to control inflammatory cascades of diseases associated with high or dysregulated levels of endogenous PLA2. Recently, varespladib was also found to inhibit snake venom PLA2 and PLA2-like toxins. Herein, ex vivo neuromuscular blocking activity assays were used to test the inhibitory activity of varespladib. The binding affinity between varespladib and a PLA2-like toxin was quantified and compared with other potential inhibitors for this class of proteins. Crystallographic and bioinformatic studies showed that varespladib binds to PrTX-I and BthTX-I into their hydrophobic channels, similarly to other previously characterized PLA2-like myotoxins. However, a new finding is that an additional varespladib binds to the MDiS region, a particular site that is related to muscle cell disruption by these toxins. The present results further advance the characterization of the molecular interactions of varespladib with PLA2-like myotoxins and provide additional evidence for this compound as a promising inhibitor candidate for different PLA2 and PLA2-like toxins.

Asunto(s)

Bothrops , Venenos de Crotálidos , Toxinas Biológicas , Animales , Humanos , Bothrops/metabolismo , Neurotoxinas , Cetoácidos , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/química , Fosfolipasas A2/química

RESUMEN

In order to address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as high affinity, their ability to penetrate dense tissues, and facility for genetic manipulation, their application in antivenoms has expanded considerably. VHHs that are active against the metalloprotease BjussuMP-II from the snake Bothrops jararacussu were selected. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. One selected clone (VHH61) showed cross-reactivity to B. brazili venom, but did not recognize the Crotalus and Lachesis genera, indicating specificity for the Bothrops genus. Through in vitro tests, the capacity to neutralize the toxicity triggered by BjussuMP-II was observed. Circular dichroism spectroscopy indicated a robust secondary structure for VHH61, and the calculated melting temperature (T M) for the clone was 56.4°C. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin's conformation and stability. The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.

Asunto(s)

Bothrops , Venenos de Crotálidos , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Animales , Antivenenos/uso terapéutico , Bothrops/metabolismo , Metaloproteasas/metabolismo , Simulación del Acoplamiento Molecular , Pruebas de Neutralización , Anticuerpos de Dominio Único/farmacología , Mordeduras de Serpientes/tratamiento farmacológico

RESUMEN

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1ß induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1ß secretion. Overall, this work helps elucidate the function of CVX in immune system cells.

Asunto(s)

Venenos de Crotálidos , Crotalus , Animales , Venenos de Crotálidos/química , Crotalus/metabolismo , Humanos , Inflamasomas , Interleucina-10 , Interleucina-1beta , Lectinas Tipo C/metabolismo , Leucocitos Mononucleares/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno

RESUMEN

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Asunto(s)

Mezclas Complejas/química , Isoformas de Proteínas/análisis , Programas Informáticos , Animales , Venenos de Crotálidos/química , Venenos de Crotálidos/genética , Crotalus/genética , Crotoxina/química , Crotoxina/genética , Fosfolipasas A2/química

RESUMEN

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

RESUMEN

Trypanosoma cruzi faces a variety of environmental scenarios during its life cycle, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible posttranslational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase TcSir2rp3 was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, known to induce reactive oxidative and nitrosactive species in the parasite, associated to that a reduction in the ROS levels was observed. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of TcSODA in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that these parasites presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at a similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible for regulating MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches, we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect to hydrogen-bonding pattern to neighbor residues, suggesting a key participation of this residue to modulate the affinity to O2- . Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection.

Asunto(s)

Mitocondrias/enzimología , Estrés Oxidativo , Sirtuinas , Trypanosoma cruzi , Oxidación-Reducción , Sirtuinas/genética , Sirtuinas/metabolismo , Superóxido Dismutasa/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética

RESUMEN

The classical nuclear import pathway is mediated by importin (Impα and Impß), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far N-terminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs.

Asunto(s)

Calorimetría/métodos , Simulación del Acoplamiento Molecular , Señales de Localización Nuclear/genética , alfa Carioferinas/genética , Transporte Activo de Núcleo Celular/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Unión Competitiva , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Ratones , Mutación , Unión Proteica , Dominios Proteicos , Electricidad Estática , alfa Carioferinas/química , alfa Carioferinas/metabolismo

RESUMEN

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10-7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound.

Asunto(s)

Anacardium/química , Ácido Gálico/farmacología , Miotoxicidad/tratamiento farmacológico , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/metabolismo , Venenos de Serpiente/enzimología , Animales , Modelos Animales de Enfermedad , Ácido Gálico/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Miotoxicidad/enzimología , Miotoxicidad/etiología , Inhibidores de Fosfolipasa A2/química , Fosfolipasas A2/química , Tallos de la Planta/química , Proteínas de Reptiles/química , Proteínas de Reptiles/metabolismo , Resonancia por Plasmón de Superficie

RESUMEN

BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.

Asunto(s)

Acetatos/farmacología , Bothrops/metabolismo , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Indoles/farmacología , Cetoácidos/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/química , Proteínas de Reptiles/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Fosfolipasas A2 Grupo II/toxicidad , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas de Reptiles/toxicidad

RESUMEN

Snakebite envenomation has been categorized by World Health Organization as a category A neglected tropical disease, since it causes chronic psychological disorders, physical disablement and death. Ophidian accidents may cause local myonecrosis that cause drastic sequelae, which are not efficiently neutralized via serum therapy. Phospholipase A2-like (PLA2-like) myotoxins have a major role in the local effects caused by several snake venoms. We previously demonstrated that chicoric acid (CA) is an efficient inhibitor of the BthTX-I myotoxin and solved the X-ray structure of complex. Herein, we assess the oligomeric behavior of the BthTX-I/CA complex in solution under different physical-chemical conditions and using toxin obtained by two different biochemical methodologies to fully elucidate structural bases of inhibition of myotoxins by CA. We demonstrated the ability of PLA2-like proteins to form different oligomeric assemblies in the presence of certain inhibitors, which can also be modulated by buffer polarity change. In the presence of ethanol, BthTX-I/CA remains predominantly in a monomeric conformation, which prevents it from being in its active form (dimeric conformation). In contrast, in the absence of ethanol, the tetramer assembly was observed, which hid key regions of the protein responsible for docking and disruption of the muscle membrane. Therefore, the "plasticity" of these proteins with regard to their abilities to form oligomeric assemblies is a key issue for the future development of therapeutic agents to complement of serum therapy.

Asunto(s)

Ácidos Cafeicos/química , Venenos de Crotálidos/química , Fosfolipasas A2/química , Multimerización de Proteína , Succinatos/química , Venenos de Crotálidos/antagonistas & inhibidores

RESUMEN

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

RESUMEN

The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT, which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results.

Asunto(s)

Bothrops , Venenos de Crotálidos/química , Regulación Alostérica , Animales , Simulación por Computador , Dispersión Dinámica de Luz , Simulación de Dinámica Molecular , Fosfolipasas A2/química , Multimerización de Proteína

RESUMEN

Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P-I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming.

Asunto(s)

Bothrops/metabolismo , Venenos de Crotálidos/enzimología , Metaloproteasas/química , Metaloproteasas/farmacología , Secuencia de Aminoácidos , Animales , Calcio/química , Cationes/química , Biología Computacional , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/farmacología , Cristalización , Cristalografía por Rayos X , Bases de Datos de Proteínas , Fibrinógeno/metabolismo , Gelatina/metabolismo , Hemorragia/enzimología , Espectrometría de Masas , Metaloproteasas/aislamiento & purificación , Ratones , Modelos Moleculares , Alineación de Secuencia , Análisis de Secuencia de Proteína , Piel/enzimología , Piel/metabolismo

RESUMEN

A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins.

RESUMEN

Envenoming by snakebite is an important global health issue that has received little attention, leading the World Health Organization to naming it as neglected tropical disease. Several snakebites present serious local symptoms manifested on victims that may not be efficiently neutralized by serum therapy. Phospholipase A2-like (PLA2-like) toxins are present in Viperidae venoms and are responsible for local myotoxic activity. Herein, we investigated the association between BthTX-I toxin and caftaric acid (CFT), a molecule present in plants. CFT neutralized neuromuscular blocking and muscle-damaging activities promoted by BthTX-I. Calorimetric and light-scattering assays demonstrated that CFT inhibitor interacted with dimeric BthTX-I. Bioinformatics simulations indicated that CFT inhibitor binds to the toxin's hydrophobic channel (HCh). According to the current myotoxic mechanism, three different regions of PLA2-like toxins have specific tasks: protein allosteric activation (HCh), membrane dockage (MDoS), and membrane rupture (MDiS). We propose CFT inhibitor interferes with the allosteric activation, which is related to the conformation change leading to the exposure/alignment of MDoS/MDiS region. This is the first report of a PLA2-like toxin fully inhibited by a compound that interacts only with its HCh region. Thus, CFT is a novel candidate to complement serum therapy and improve the treatment of snakebite.

Asunto(s)

Venenos de Crotálidos/toxicidad , Miotoxicidad/tratamiento farmacológico , Bloqueantes Neuromusculares/toxicidad , Fenoles/farmacología , Fosfolipasas A2/química , Animales , Masculino , Ratones , Miotoxicidad/etiología , Fosfolipasas A2/metabolismo , Conformación Proteica