RESUMO
Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Venenos de Aranha/química , Animais , Proteínas de Artrópodes/análise , Austrália , Dípteros/efeitos dos fármacos , Dissulfetos , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Filogenia , Conformação Proteica , Proteômica/métodos , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Aranhas/genéticaRESUMO
Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus, induces pain in mice by inhibiting inactivation of voltage-gated sodium (NaV) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach NaV channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence.
Assuntos
Evolução Molecular , Neurotoxinas/genética , Poliaminas/química , Aranhas/genética , Sequência de Aminoácidos/genética , Animais , Austrália , Sequência Conservada/genética , Feminino , Humanos , Masculino , Camundongos , Neurotoxinas/química , Neurotoxinas/metabolismo , Peptídeos/genética , Filogenia , Poliaminas/metabolismo , Comportamento Sexual Animal/fisiologia , Venenos de Aranha/genética , Aranhas/patogenicidade , Transcriptoma/genética , Vertebrados/genética , Vertebrados/fisiologiaRESUMO
Most neurodegenerative disorders take decades to develop, and their early detection is challenged by confounding non-pathological ageing processes. Therefore, the discovery of genes and molecular pathways in both peripheral and brain tissues that are highly predictive of disease evolution is necessary. To find genes that influence Alzheimer's disease (AD) and Parkinson's disease (PD) pathogenesis, human RNA-Seq transcriptomic data from Brodmann Area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC), whole blood (WB), and peripheral blood mononuclear cells (PBMC) were analysed using a combination of differential gene expression and a random forest-based machine learning algorithm. The results suggest that there is little overlap between PD and AD, and the AD brain signature is unique mainly compared to blood-based samples. Moreover, the AD-BA9 was characterised by changes in 'nervous system development' with Myocyte-specific enhancer factor 2C (Mef2C), encoding a transcription factor that induces microglia activation, a prominent feature. The peripheral AD transcriptome was associated with alterations in 'viral process', and FYN, which has been previously shown to link amyloid-beta and tau, was the prominent feature. However, in the absence of any overlap with the central transcriptome, it is unclear whether peripheral FYN levels reflect AD severity or progression. In PD, central and peripheral signatures are characterised by anomalies in 'exocytosis' and specific genes related to the SNARE complex, including Vesicle-associated membrane protein 2 (VAMP2), Syntaxin 1A (STX1A), and p21-activated kinase 1 (PAK1). This is consistent with our current understanding of the physiological role of alpha-synuclein and how alpha-synuclein oligomers compromise vesicle docking and neurotransmission. Overall, the results describe distinct disease-specific pathomechanisms, both within the brain and peripherally, for the two most common neurodegenerative disorders.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Parkinson , Doença de Alzheimer/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Doença de Parkinson/metabolismo , Transcriptoma , alfa-Sinucleína/metabolismoRESUMO
Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus); although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less diversifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.
Assuntos
Venenos de Aranha , Aranhas , Animais , Humanos , Venenos de Aranha/toxicidade , Venenos de Aranha/química , Árvores , Austrália , PeptídeosRESUMO
Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.
Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/administração & dosagem , Canais Iônicos Sensíveis a Ácido/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Venenos de Aranha/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Bloqueadores do Canal Iônico Sensível a Ácido/química , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/metabolismoRESUMO
Summary: ArachnoServer is a manually curated database that consolidates information on the sequence, structure, function and pharmacology of spider-venom toxins. Although spider venoms are complex chemical arsenals, the primary constituents are small disulfide-bridged peptides that target neuronal ion channels and receptors. Due to their high potency and selectivity, these peptides have been developed as pharmacological tools, bioinsecticides and drug leads. A new version of ArachnoServer (v3.0) has been developed that includes a bioinformatics pipeline for automated detection and analysis of peptide toxin transcripts in assembled venom-gland transcriptomes. ArachnoServer v3.0 was updated with the latest sequence, structure and functional data, the search-by-mass feature has been enhanced, and toxin cards provide additional information about each mature toxin. Availability and implementation: http://arachnoserver.org. Contact: support@arachnoserver.org. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Venenos de Aranha/química , Animais , Automação Laboratorial , Dissulfetos/química , Proteínas de Insetos/química , Peptídeos/química , Venenos de Aranha/análiseRESUMO
Despite the staggering diversity of venomous animals, there seems to be remarkable convergence in regard to the types of proteins used as toxin scaffolds. However, our understanding of this fascinating area of evolution has been hampered by the narrow taxonomical range studied, with entire groups of venomous animals remaining almost completely unstudied. One such group is centipedes, class Chilopoda, which emerged about 440 Ma and may represent the oldest terrestrial venomous lineage next to scorpions. Here, we provide the first comprehensive insight into the chilopod "venome" and its evolution, which has revealed novel and convergent toxin recruitments as well as entirely new toxin families among both high- and low molecular weight venom components. The ancient evolutionary history of centipedes is also apparent from the differences between the Scolopendromorpha and Scutigeromorpha venoms, which diverged over 430 Ma, and appear to employ substantially different venom strategies. The presence of a wide range of novel proteins and peptides in centipede venoms highlights these animals as a rich source of novel bioactive molecules. Understanding the evolutionary processes behind these ancient venom systems will not only broaden our understanding of which traits make proteins and peptides amenable to neofunctionalization but it may also aid in directing bioprospecting efforts.
Assuntos
Venenos de Artrópodes/metabolismo , Artrópodes/classificação , Artrópodes/metabolismo , Evolução Molecular , Animais , Venenos de Artrópodes/genética , Genômica/métodos , Família Multigênica , FilogeniaRESUMO
BACKGROUND: Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. RESULTS: Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. CONCLUSIONS: Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology.
Assuntos
Família Multigênica , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Austrália , Códon , Sequência Conservada , Evolução Molecular , Feminino , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Filogenia , Matrizes de Pontuação de Posição Específica , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Alinhamento de Sequência , Venenos de Aranha/química , Aranhas/classificação , Aranhas/genéticaRESUMO
More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.
Assuntos
Sequenciamento por Nanoporos , Alelos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Repetições de Microssatélites/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Short tandem repeat (STR) expansion disorders are an important cause of human neurological disease. They have an established role in more than 40 different phenotypes including the myotonic dystrophies, Fragile X syndrome, Huntington's disease, the hereditary cerebellar ataxias, amyotrophic lateral sclerosis and frontotemporal dementia. MAIN BODY: STR expansions are difficult to detect and may explain unsolved diseases, as highlighted by recent findings including: the discovery of a biallelic intronic 'AAGGG' repeat in RFC1 as the cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS); and the finding of 'CGG' repeat expansions in NOTCH2NLC as the cause of neuronal intranuclear inclusion disease and a range of clinical phenotypes. However, established laboratory techniques for diagnosis of repeat expansions (repeat-primed PCR and Southern blot) are cumbersome, low-throughput and poorly suited to parallel analysis of multiple gene regions. While next generation sequencing (NGS) has been increasingly used, established short-read NGS platforms (e.g., Illumina) are unable to genotype large and/or complex repeat expansions. Long-read sequencing platforms recently developed by Oxford Nanopore Technology and Pacific Biosciences promise to overcome these limitations to deliver enhanced diagnosis of repeat expansion disorders in a rapid and cost-effective fashion. CONCLUSION: We anticipate that long-read sequencing will rapidly transform the detection of short tandem repeat expansion disorders for both clinical diagnosis and gene discovery.
Assuntos
Expansão das Repetições de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Repetições de Microssatélites/genética , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Análise de Sequência de DNA/métodos , Animais , HumanosRESUMO
A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
Assuntos
Venenos de Formiga/química , Formigas/metabolismo , Perfilação da Expressão Gênica , Proteínas de Insetos/análise , Neurotoxinas/análise , Proteoma , Proteômica , Transcriptoma , Animais , Venenos de Formiga/genética , Venenos de Formiga/toxicidade , Formigas/genética , Cálcio/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Bases de Dados Genéticas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/toxicidade , Camundongos Endogâmicos C57BL , Neurotoxinas/genética , Neurotoxinas/toxicidade , Espectrometria de Massas em TandemRESUMO
Consistent with their diverse pharmacology, peptides derived from venomous animals have been developed as drugs to treat disorders as diverse as hypertension, diabetes and chronic pain. Melanoma has a poor prognosis due in part to its metastatic capacity, warranting further development of novel targeted therapies. This prompted us to examine the anti-melanoma activity of the spider peptides gomesin (AgGom) and a gomesin-like homolog (HiGom). AgGom and HiGom dose-dependently reduced the viability and proliferation of melanoma cells whereas it had no deleterious effects on non-transformed neonatal foreskin fibroblasts. Concordantly, gomesin-treated melanoma cells showed a reduced G0/G1 cell population. AgGom and HiGom compromised proliferation of melanoma cells via activation of the p53/p21 cell cycle check-point axis and the Hippo signaling cascade, together with attenuation of the MAP kinase pathway. We show that both gomesin peptides exhibit antitumoral activity in melanoma AVATAR-zebrafish xenograft tumors and that HiGom also reduces tumour progression in a melanoma xenograft mouse model. Taken together, our data highlight the potential of gomesin for development as a novel melanoma-targeted therapy.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Melanoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Xenoenxertos , Camundongos , Transplante de Neoplasias , Resultado do Tratamento , Peixe-ZebraRESUMO
The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.
RESUMO
The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease.
RESUMO
UNLABELLED: Sepiadarium austrinum, the southern bottletail squid, is a small squid that inhabits soft sediments along Australia's south-east coast. When provoked, it rapidly secretes large volumes of slime, presumably as a form of chemical defense. We analyzed the proteomic composition of this slime using tandem mass spectrometry and transcriptomics and found that it was remarkably complex with 1735 identified protein groups (FDR:0.01). To investigate the chemical defense hypothesis we performed an Artemia toxicity assay and used sequence analysis to search for toxin-like molecules. Although the slime did not appear to be toxic to Artemia we found 13 proteins in slime with the hallmarks of toxins, namely cysteine richness, short length, a signal peptide and/or homology to known toxins. These included three short (80-130AA) cysteine rich secreted proteins with no homology to proteins on the NCBI or UniProt databases. Other protein families found included, CAP, phospholipase-B, ShKT-like peptides, peptidase S10, Kunitz BPTI and DNase II. Quantitative analysis using intensity based absolute quantification (iBAQ via MaxQuant) revealed 20 highly abundant proteins, accounting for 67% of iBAQ signal, and three of these were toxin-like. No mucin homologues were found suggesting that the structure of the slime gel may be formed by an unknown mechanism. BIOLOGICAL SIGNIFICANCE: This study is the first known instance of a slime secretion from a cephalopod to be analyzed by proteomics methods and is the first investigation of a member of the family Sepiadariidae using proteomic methods. 1735 proteins were identified with 13 of these fitting criteria established for the identification of putative toxins. The slime is dominated by 20 highly abundant proteins with secreted, cysteine rich proteins. The study highlights the importance of 'omics approaches in understanding novel organisms.
Assuntos
Decapodiformes/química , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Animais , Decapodiformes/imunologia , Géis/química , Proteínas/metabolismo , Proteoma/análise , Proteoma/toxicidadeRESUMO
Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.
Assuntos
Proteínas de Artrópodes/isolamento & purificação , Dípteros , Inseticidas/isolamento & purificação , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/toxicidade , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Alinhamento de Sequência , Análise de Sequência de Proteína , Ovinos/parasitologia , Testes de Toxicidade , TranscriptomaRESUMO
Over a period of more than 300 million years, spiders have evolved complex venoms containing an extraordinary array of toxins for prey capture and defense against predators. The major components of most spider venoms are small disulfide-bridged peptides that are highly stable and resistant to proteolytic degradation. Moreover, many of these peptides have high specificity and potency toward molecular targets of therapeutic importance. This unique combination of bioactivity and stability has made spider-venom peptides valuable both as pharmacological tools and as leads for drug development. This review describes recent advances in spider-venom-based drug discovery pipelines. We discuss spider-venom-derived peptides that are currently under investigation for treatment of a diverse range of pathologies including pain, stroke and cancer.
Assuntos
Descoberta de Drogas , Venenos de Aranha/química , Animais , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (â¼132kDa) with â¼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.