RESUMO
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/ultraestrutura , Peptídeos/metabolismo , Venenos de Aranha/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Ativação do Canal Iônico , Peptídeos/toxicidade , Domínios Proteicos , Venenos de Aranha/toxicidade , Aranhas , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Venenos de Aranha/química , Substituição de Aminoácidos , Animais , Humanos , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células Sf9 , SpodopteraRESUMO
Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here, we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high-avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency. The "double-knot" toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.
Assuntos
Venenos de Aranha/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Ratos , Venenos de Aranha/química , Canais de Cátion TRPV/química , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismo , Proteínas de Xenopus/químicaRESUMO
The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.
Assuntos
Nociceptores/efeitos dos fármacos , Papio/metabolismo , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Aranhas/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Gânglios Espinais/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Canais Iônicos/metabolismo , Camundongos , Dor/tratamento farmacológico , Tetrodotoxina/farmacologiaRESUMO
Nasopharyngeal carcinoma (NPC) is a prevalent malignancy that usually occurs among the nose and throat. Due to mild initial symptoms, most patients are diagnosed in the late stage, and the recurrence rate of tumors is high, resulting in many deaths every year. Traditional radiotherapy and chemotherapy are prone to causing drug resistance and significant side effects. Therefore, searching for new bioactive drugs including anticancer peptides is necessary and urgent. LVTX-8 is a peptide toxin synthesized from the cDNA library of the spider Lycosa vittata, which is consisting of 25 amino acids. In this study, a series of in vitro cell experiments such as cell toxicity, colony formation, and cell migration assays were performed to exam the anticancer activity of LVTX-8 in NPC cells (5-8F and CNE-2). The results suggested that LVTX-8 significantly inhibited cell proliferation and migration of NPC cells. To find the potential molecular targets for the anticancer capability of LVTX-8, high-throughput proteomic and bioinformatics analysis were conducted on NPC cells. The results identified EXOSC1 as a potential target protein with significantly differential expression levels under LVTX-8+/LVTX-8- conditions. The results in this research indicate that spider peptide toxin LVTX-8 exhibits significant anticancer activity in NPC, and EXOSC1 may serve as a target protein for its anticancer activity. These findings provide a reference for the development of new therapeutic drugs for NPC and offer new ideas for the discovery of biomarkers related to NPC diagnosis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the data set identifier PXD050542.
Assuntos
Antineoplásicos , Movimento Celular , Proliferação de Células , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proteômica , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Proteômica/métodos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Venenos de Aranha/farmacologia , Venenos de Aranha/química , Animais , Peptídeos/farmacologia , Peptídeos/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
A special category of phospholipase D (PLD) in the venom of the brown recluse spider (Loxosceles reclusa) and several other sicariid spiders accounts for the dermonecrosis and many of the other clinical symptoms of envenomation. Related proteins are produced by other organisms, including fungi and bacteria. These PLDs are often referred to as sphingomyelinase Ds (SMase Ds) because they cleave sphingomyelin (SM) to choline and "ceramide phosphate." The lipid product has actually been found to be a novel sphingolipid: ceramide 1,3-cyclic phosphate (Cer1,3P). Since there are no effective treatments for the injury induced by the bites of these spiders, SMase D/PLDs are attractive targets for therapeutic intervention, and some of their features will be described in this minireview. In addition, two simple methods are described for detecting the characteristic SMase D activity using a fluorescent SM analog, (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-SM (C12-NBD-SM), that is cleaved to C12-NBD-Cer1,3P, which is easily separated from other potential metabolites by thin-layer chromatography and visualized under UV light. Besides confirming that C12-NBD-Cer1,3P is the only product detected upon incubation of C12-NBD-SM with brown recluse spider venom, the method was also able to detect for the first time very low levels of activity in venom from another spider, Kukulcania hibernalis The simplicity of the methods makes it relatively easy to determine this signature activity of SMase D/PLD. SIGNIFICANCE STATEMENT: The sphingomyelinase D/phospholipase D that are present in the venom of the brown recluse spider and other sources cause considerable human injury, but detection of the novel sphingolipid product, ceramide 1,3-cyclic phosphate, is not easy by previously published methods. This minireview describes simple methods for detection of this activity that will be useful for studies of its occurrence in spider venoms and other biological samples, perhaps including lesions from suspected spider bites and infections.
Assuntos
Fosfolipase D , Venenos de Aranha , Aranhas , Humanos , Animais , Esfingomielina Fosfodiesterase , Fosfolipase D/química , Fosfolipase D/metabolismo , Ceramidas , Fosfatos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Aranhas/metabolismoRESUMO
Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The specific mechanism through which excessive load induces TMJ osteoarthritis is not fully understood; however, mechanically-activated (MA) ion channels play a crucial role. Among these channels, Piezo1 has been identified as a mediator of chondrocyte catabolic responses and is markedly increased in osteoarthritis. Our observations indicate that, under excessive load conditions, endoplasmic reticulum stress in chondrocytes results in apoptosis of the TMJ chondrocytes. Importantly, using the Piezo1 inhibitor GsMTx4 demonstrates its potential to alleviate this condition. Furthermore, Piezo1 mediates endoplasmic reticulum stress in chondrocytes by inducing calcium ion influx. Our research substantiates the role of Piezo1 as a pivotal ion channel in mediating chondrocyte overload. It elucidates the link between excessive load, cell apoptosis, and calcium ion influx through Piezo1. The findings underscore Piezo1 as a key player in the pathogenesis of TMJ osteoarthritis, shedding light on potential therapeutic interventions for this condition.
Assuntos
Apoptose , Cálcio , Condrócitos , Estresse do Retículo Endoplasmático , Canais Iônicos , Osteoartrite , Articulação Temporomandibular , Condrócitos/metabolismo , Condrócitos/patologia , Canais Iônicos/metabolismo , Canais Iônicos/genética , Animais , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia , Cálcio/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Humanos , Camundongos , Transdução de Sinais , Venenos de Aranha , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
The spider venom protein, double-knot toxin (DkTx), partitions into the cellular membrane and binds bivalently to the pain-sensing ion channel, TRPV1, triggering long-lasting channel activation. In contrast, its monovalent single knots membrane partition poorly and invoke rapidly reversible TRPV1 activation. To discern the contributions of the bivalency and membrane affinity of DkTx to its sustained mode of action, here, we developed diverse toxin variants including those containing truncated linkers between individual knots, precluding bivalent binding. Additionally, by appending the single-knot domains to the Kv2.1 channel-targeting toxin, SGTx, we created monovalent double-knot proteins that demonstrated higher membrane affinity and more sustained TRPV1 activation than the single-knots. We also produced hyper-membrane affinity-possessing tetra-knot proteins, (DkTx)2 and DkTx-(SGTx)2, that demonstrated longer-lasting TRPV1 activation than DkTx, establishing the central role of the membrane affinity of DkTx in endowing it with its sustained TRPV1 activation properties. These results suggest that high membrane affinity-possessing TRPV1 agonists can potentially serve as long-acting analgesics.
Assuntos
Membrana Celular , Venenos de Aranha , Canais de Cátion TRPV , Membrana Celular/metabolismo , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Animais , Canais de Cátion TRPV/metabolismo , Dor/metabolismo , Ligação Proteica , Analgésicos , Transporte de ÍonsRESUMO
Bronchial airways and lung parenchyma undergo both static and dynamic stretch in response to normal breathing as well as in the context of insults such as mechanical ventilation (MV) or in diseases such as asthma and chronic obstructive pulmonary disease (COPD) which lead to airway remodeling involving increased extracellular matrix (ECM) production. Here, the role of fibroblasts is critical, but the relationship between stretch- and fibroblast-induced ECM remodeling under these conditions is not well-explored. Piezo (PZ) channels play a role in mechanotransduction in many cell and organ systems, but their role in mechanical stretch-induced airway remodeling is not known. To explore this, we exposed human lung fibroblasts to 10% static stretch on a background of 5% oscillations for 48 h, with no static stretch considered controls. Collagen I, fibronectin, alpha-smooth muscle actin (α-SMA), and Piezo 1 (PZ1) expression was determined in the presence or absence of Yoda1 (PZ1 agonist) or GsMTx4 (PZ1 inhibitor). Collagen I, fibronectin, and α-SMA expression was increased by stretch and Yoda1, whereas pretreatment with GsMTx4 or knockdown of PZ1 by siRNA blunted this effect. Acute stretch in the presence and absence of Yoda1 demonstrated activation of the ERK pathway but not Smad. Measurement of [Ca2+]i responses to histamine showed significantly greater responses following stretch, effects that were blunted by knockdown of PZ1. Our findings identify an essential role for PZ1 in mechanical stretch-induced production of ECM mediated by ERK phosphorylation and Ca2+ influx in lung fibroblasts. Targeting PZ channels in fibroblasts may constitute a novel approach to ameliorate airway remodeling by decreasing ECM deposition.NEW & NOTEWORTHY The lung is an inherently mechanosensitive organ that can respond to mechanical forces in adaptive or maladaptive ways, including via remodeling resulting in increased fibrosis. We explored the mechanisms that link mechanical forces to remodeling using human lung fibroblasts. We found that mechanosensitive Piezo channels increase with stretch and mediate extracellular matrix formation and the fibroblast-to-myofibroblast transition that occurs with stretch. Our data highlight the importance of Piezo channels in lung mechanotransduction toward remodeling.
Assuntos
Fibroblastos , Canais Iônicos , Pulmão , Mecanotransdução Celular , Humanos , Pulmão/metabolismo , Pulmão/citologia , Fibroblastos/metabolismo , Canais Iônicos/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Remodelação das Vias Aéreas , Actinas/metabolismo , Células Cultivadas , Estresse Mecânico , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cálcio/metabolismo , Venenos de Aranha , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Piezo1 functions as a special transducer of mechanostress into electrochemical signals and is implicated in the pathogenesis of various diseases across different disciplines. However, whether Piezo1 contributes to the pathogenesis of lupus nephritis (LN) remains elusive. To study this, we applied an agonist and antagonist of Piezo1 to treat lupus-prone MRL/lpr mice. Additionally, a podocyte-specific Piezo1 knockout mouse model was also generated to substantiate the role of Piezo1 in podocyte injury induced by pristane, a murine model of LN. A marked upregulation of Piezo1 was found in podocytes in both human and murine LN. The Piezo1 antagonist, GsMTx4, significantly alleviated glomerulonephritis and tubulointerstitial damage, improved kidney function, decreased proteinuria, and mitigated podocyte foot process effacement in MRL/lpr mice. Moreover, podocyte-specific Piezo1 deletion showed protective effects on the progression of proteinuria and podocyte foot process effacement in the murine LN model. Mechanistically, Piezo1 expression was upregulated by inflammatory cytokines (IL-6, TNF-α and IFN-γ), soluble urokinase Plasminogen Activator Receptor and its own activation. Activation of Piezo1 elicited calcium influx, which subsequently enhanced Rac1 activity and increased active paxillin, thereby promoting cytoskeleton remodeling and decreasing podocyte motility. Thus, our work demonstrated that Piezo1 contributed to podocyte injury and proteinuria progression in LN. Hence, targeted therapy aimed at decreasing or inhibiting Piezo1 could represent a novel strategy to treat LN.
Assuntos
Citoesqueleto , Modelos Animais de Doenças , Canais Iônicos , Nefrite Lúpica , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Podócitos , Proteinúria , Animais , Podócitos/patologia , Podócitos/metabolismo , Nefrite Lúpica/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/genética , Humanos , Proteinúria/genética , Proteinúria/patologia , Proteinúria/metabolismo , Proteinúria/etiologia , Canais Iônicos/metabolismo , Canais Iônicos/genética , Camundongos , Citoesqueleto/metabolismo , Feminino , Venenos de Aranha/farmacologia , Mecanotransdução Celular , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Adulto , Masculino , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/farmacologia , Aminoácidos/química , Canais Iônicos Sensíveis a Ácido/genética , Células HEK293 , Humanos , Peptídeos/química , Venenos de Aranha/química , TransfecçãoRESUMO
Agricultural crops are targeted by various pathogens (fungi, bacteria, and viruses) and pests (herbivorous arthropods). Antimicrobial and insecticidal peptides are increasingly recognized as eco-friendly tools for crop protection due to their low propensity for resistance development and the fact that they are fully biodegradable. However, historical challenges have hindered their development, including poor stability, limited availability, reproducibility issues, high production costs, and unwanted toxicity. Toxicity is a primary concern because crop-protective peptides interact with various organisms of environmental and economic significance. This review focuses on the potential of genetically encoded peptide libraries like the use of two-hybrid-based methods for antimicrobial peptides identification and insecticidal spider venom peptides as two main approaches for targeting plant pathogens and pests. We discuss some key findings and challenges regarding the practical application of each strategy. We conclude that genetically encoded peptide library- and spider venom-derived crop protective peptides offer a sustainable and environmentally responsible approach for addressing modern crop protection needs in the agricultural sector.
Assuntos
Produtos Agrícolas , Biblioteca de Peptídeos , Venenos de Aranha , Venenos de Aranha/química , Venenos de Aranha/genética , Inseticidas/química , Inseticidas/farmacologia , Animais , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Proteção de Cultivos/métodosRESUMO
AIMS: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. METHODS: To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. RESULTS: The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. CONCLUSION: These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.
Assuntos
Canais Iônicos , Lipopolissacarídeos , Osteoblastos , Osteoprotegerina , Porphyromonas gingivalis , Ligante RANK , Estresse Mecânico , Animais , Camundongos , Ligante RANK/metabolismo , Lipopolissacarídeos/farmacologia , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoprotegerina/metabolismo , Canais Iônicos/metabolismo , RNA Interferente Pequeno , Fosforilação , Venenos de Aranha , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
AIM: Tarantulas are one of the largest predatory arthropods in tropical regions. Tarantulas though not lethal to humans, their venomous bite kills small animals and insect upon which they prey. To understand the abiotic and biotic components involved in Neotropical tarantula bites, we conducted a venom-microbiomics study in eight species from Costa Rica. METHODS AND RESULTS: We determined that the toxin profiles of tarantula venom are highly diverse using shotgun proteomics; the most frequently encountered toxins were ω-Ap2 toxin, neprilysin-1, and several teraphotoxins. Through culture-independent and culture-dependent methods, we determined the microbiota present in the venom and excreta to evaluate the presence of pathogens that could contribute to primary infections in animals, including humans. The presence of opportunistic pathogens with hemolytic activity was observed, with a prominence of Stenotrophomonas in the venoms. Other bacteria found in venoms and excreta with hemolytic activity included members of the genera Serratia, Bacillus, Acinetobacter, Microbacterium, and Morganella. CONCLUSIONS: Our data shed light on the venom- and gut-microbiome associated with Neotropical tarantulas. This information may be useful for treating bites from these arthropods in both humans and farm animals, while also providing insight into the toxins and biodiversity of this little-explored microenvironment.
Assuntos
Venenos de Aranha , Aranhas , Animais , Aranhas/microbiologia , Costa Rica , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/genética , Proteômica , Microbioma Gastrointestinal , MicrobiotaRESUMO
Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays.
Assuntos
Esfingomielina Fosfodiesterase , Picada de Aranha , Venenos de Aranha , Humanos , Esfingomielina Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/toxicidade , Rim , Morte CelularRESUMO
Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.
Assuntos
Venenos de Artrópodes/química , Proteínas de Insetos/química , Lepidópteros/química , Neuropeptídeos/química , Dor/genética , Animais , Venenos de Artrópodes/genética , Evolução Molecular , Proteínas de Insetos/genética , Mariposas/química , Neuropeptídeos/genética , Peptídeos/química , Peptídeos/genética , Proteômica , Venenos de Aranha/química , Venenos de Aranha/genética , Transcriptoma/genéticaRESUMO
Ion channels on cell membrane are molecular targets of more than half peptide neurotoxins from spiders. From Pardosa pseudoannulata, a predatory spider on a range of insect pests, we characterized a peptide neurotoxin PPTX-04 with an insecticidal activity. PPTX-04 showed high toxicity to Nilaparvata lugens, a main prey of P. pseudoannulata, and the toxicity was not affected by the resistance to etofenprox (IUPAC chemical name:1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl)methoxy]propan-2-yl]benzene, purity: 99%). On N. lugens voltage-gated sodium channel NlNav1 expressed in Xenopus oocytes, PPTX-04 prolonged the channel opening and induced tail currents, which is similar to pyrethroid insecticides. However, PPTX-04 potency on NlNav1 was not affected by mutations conferring pyrethroid resistance in insects, which revealed that PPTX-04 and pyrethroids should act on different receptors in NlNav1. In contrast, two mutations at the extracellular site 4 significantly reduced PPTX-04 potency, which indicated that PPTX-04 would act on a potential receptor containing the site 4 in NlNav1. The result from the molecular docking supported the conclusion that the binding pocket of PPTX-04 in NlNav1 should contain the site 4. In summary, PPTX-04 had high insecticidal activity through acting on a distinct receptor site in insect Nav, and was a potential resource to control insect pests and manage resistance to pyrethroids.
Assuntos
Inseticidas , Neurotoxinas , Venenos de Aranha , Aranhas , Canais de Sódio Disparados por Voltagem , Animais , Inseticidas/farmacologia , Inseticidas/química , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Venenos de Aranha/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Neurotoxinas/farmacologia , Neurotoxinas/toxicidade , Piretrinas/farmacologia , Hemípteros/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Xenopus laevis , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/químicaRESUMO
The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit ß (ATP SSß) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSß of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.
Assuntos
Peptídeos , Venenos de Aranha , Animais , Venenos de Aranha/química , Venenos de Aranha/genética , Peptídeos/farmacologia , Peptídeos/química , Ácaros/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Tetranychidae/efeitos dos fármacos , Tetranychidae/genética , Controle Biológico de Vetores/métodos , Sequência de Aminoácidos , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/química , Comportamento Predatório/efeitos dos fármacosRESUMO
BACKGROUND: Spiders comprise a hyperdiverse lineage of predators with venom systems, yet the origin of functionally novel spider venom glands remains unclear. Previous studies have hypothesized that spider venom glands originated from salivary glands or evolved from silk-producing glands present in early chelicerates. However, there is insufficient molecular evidence to indicate similarity among them. Here, we provide comparative analyses of genome and transcriptome data from various lineages of spiders and other arthropods to advance our understanding of spider venom gland evolution. RESULTS: We generated a chromosome-level genome assembly of a model spider species, the common house spider (Parasteatoda tepidariorum). Module preservation, GO semantic similarity, and differentially upregulated gene similarity analyses demonstrated a lower similarity in gene expressions between the venom glands and salivary glands compared to the silk glands, which questions the validity of the salivary gland origin hypothesis but unexpectedly prefers to support the ancestral silk gland origin hypothesis. The conserved core network in the venom and silk glands was mainly correlated with transcription regulation, protein modification, transport, and signal transduction pathways. At the genetic level, we found that many genes in the venom gland-specific transcription modules show positive selection and upregulated expressions, suggesting that genetic variation plays an important role in the evolution of venom glands. CONCLUSIONS: This research implies the unique origin and evolutionary path of spider venom glands and provides a basis for understanding the diverse molecular characteristics of venom systems.
Assuntos
Artrópodes , Venenos de Aranha , Animais , Transcriptoma , Venenos de Aranha/genética , Evolução Molecular , Genômica , Artrópodes/genética , Glândulas Salivares/metabolismo , Seda/genética , Seda/metabolismo , FilogeniaRESUMO
PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund's adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.