RESUMO
Progressive articular surface degradation during arthritis causes ongoing pain and hyperalgesia that lead to the development of functional disability. TRPA1 channel significantly contributes to the activation of sensory neurons that initiate neurogenic inflammation and mediates pain signal transduction to the central nervous system. Peptide Ms 9a-1 from the sea anemone Metridium senile is a positive allosteric modulator of TRPA1 and shows significant anti-inflammatory and analgesic activity in different models of pain. We used a model of monosodium iodoacetate (MIA)-induced osteoarthritis to evaluate the anti-inflammatory properties of Ms 9a-1 in comparison with APHC3 (a polypeptide modulator of TRPV1 channel) and non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and ibuprofen. Administration of Ms 9a-1 (0.1 mg/kg, subcutaneously) significantly reversed joint swelling, disability, thermal and mechanical hypersensitivity, and grip strength impairment. The effect of Ms 9a-1 was equal to or better than that of reference drugs. Post-treatment histological analysis revealed that long-term administration of Ms9a-1 could reduce inflammatory changes in joints and prevent the progression of cartilage and bone destruction at the same level as meloxicam. Peptide Ms 9a-1 showed significant analgesic and anti-inflammatory effects in the model of MIA-induced OA, and therefore positive allosteric modulators could be considered for the alleviation of OA symptoms.
Assuntos
Osteoartrite , Anêmonas-do-Mar , Animais , Meloxicam/efeitos adversos , Modelos Animais de Doenças , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Dor , Anti-Inflamatórios/efeitos adversos , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Peptídeos/uso terapêutico , Ácido Iodoacético/toxicidadeRESUMO
Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.
Assuntos
Miniproteínas Nó de Cistina , Venenos de Aranha , Aranhas , Toxinas Biológicas , Ratos , Animais , Canal de Cátion TRPA1/genética , Aranhas/química , Neurotoxinas , Espectroscopia de Ressonância Magnética , Dissulfetos , Venenos de Aranha/farmacologia , Venenos de Aranha/químicaRESUMO
Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund's adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins.
Assuntos
Ansiolíticos , Proteína HMGB3 , Anêmonas-do-Mar , Toxinas Biológicas , Ratos , Camundongos , Humanos , Animais , Ansiolíticos/farmacologia , Anêmonas-do-Mar/química , Diclofenaco , Proteína HMGB2 , Peptídeos/farmacologia , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Toxinas Biológicas/farmacologia , Fatores de Transcrição , Roedores , Anti-Inflamatórios/farmacologiaRESUMO
Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.
Assuntos
Receptores Purinérgicos P2X7 , Anêmonas-do-Mar , Animais , Anêmonas-do-Mar/metabolismo , Simulação de Acoplamento Molecular , Peptídeos/química , Trifosfato de Adenosina/metabolismoRESUMO
Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.
Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Doença de Parkinson , Anêmonas-do-Mar , Humanos , Animais , Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/genética , Tirosina 3-Mono-Oxigenase , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genéticaRESUMO
The TRPA1 channel is involved in a variety of physiological processes and its activation leads to pain perception and the development of inflammation. Peptide Ms 9a-1 from sea anemone Metridium senile is a positive modulator of TRPA1 and causes significant analgesic and anti-inflammatory effects by desensitization of TRPA1-expressing sensory neurons. For structural and functional analysis of Ms 9a-1, we produced four peptides-Ms 9a-1 without C-terminal domain (abbreviated as N-Ms), short C-terminal domain Ms 9a-1 alone (C-Ms), and two homologous peptides (Ms 9a-2 and Ms 9a-3). All tested peptides possessed a reduced potentiating effect on TRPA1 compared to Ms 9a-1 in vitro. None of the peptides reproduced analgesic and anti-inflammatory properties of Ms 9a-1 in vivo. Peptides N-Ms and C-Ms were able to reduce pain induced by AITC (selective TRPA1 agonist) but did not decrease AITC-induced paw edema development. Fragments of Ms 9a-1 did not effectively reverse CFA-induced thermal hyperalgesia and paw edema. Ms 9a-2 and Ms 9a-3 possessed significant effects and anti-inflammatory properties in some doses, but their unexpected efficacy and bell-shape dose-responses support the hypothesis of other targets involved in their effects in vivo. Therefore, activity comparison of Ms 9a-1 fragments and homologues peptides revealed structural determinants important for TRPA1 modulation, as well as analgesic and anti-inflammatory properties of Ms9a-1.
Assuntos
Analgésicos , Anêmonas-do-Mar , Analgésicos/química , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Edema/induzido quimicamente , Edema/tratamento farmacológico , Proteínas do Olho , Fragmentos de Peptídeos , Peptídeos/química , Canal de Cátion TRPA1RESUMO
Human neuroblastoma SH-SY5Y is a prominent neurobiological tool used for studying neuropathophysiological processes. We investigated acid-sensing (ASIC) and transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) ion channels present in untreated and differentiated neuroblastoma SH-SY5Y to propose a new means for their study in neuronal-like cells. Using a quantitative real-time PCR and a whole-cell patch-clamp technique, ion channel expression profiles, functionality, and the pharmacological actions of their ligands were characterized. A low-level expression of ASIC1a and ASIC2 was detected in untreated cells. The treatment with 10 µM of retinoic acid (RA) for 6 days resulted in neuronal differentiation that was accompanied by a remarkable increase in ASIC1a expression, while ASIC2 expression remained almost unaltered. In response to acid stimuli, differentiated cells showed prominent ASIC-like currents. Detailed kinetic and pharmacological characterization suggests that homomeric ASIC1a is a dominant isoform among the present ASIC channels. RA-treatment also reduced the expression of TRPV1 and TRPA1, and minor electrophysiological responses to their agonists were found in untreated cells. Neuroblastoma SH-SY5Y treated with RA can serve as a model system to study the effects of different ligands on native human ASIC1a in neuronal-like cells. This approach can improve the characterization of modulators for the development of new neuroprotective and analgesic drugs.
RESUMO
Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1-Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1-Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1-Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.
Assuntos
Antagonistas Nicotínicos , Peptídeos , Receptores Nicotínicos , Anêmonas-do-Mar , Animais , Ratos , Cistina , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/isolamento & purificação , Antagonistas Nicotínicos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Receptores Nicotínicos/metabolismo , Anêmonas-do-Mar/químicaRESUMO
Arthritis is a widespread inflammatory disease associated with progressive articular surface degradation, ongoing pain, and hyperalgesia causing the development of functional limitations and disability. TRPV1 channel is one of the high-potential targets for the treatment of inflammatory diseases. Polypeptide APHC3 from sea anemone Heteractis crispa is a mode-selective TRPV1 antagonist that causes mild hypothermia and shows significant anti-inflammatory and analgesic activity in different models of pain. We evaluated the anti-inflammatory properties of APHC3 in models of monosodium iodoacetate (MIA)-induced osteoarthritis and complete Freund's adjuvant (CFA)-induced rheumatoid monoarthritis in comparison with commonly used non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, and meloxicam. Subcutaneous administration of APHC3 (0.1 mg/kg) significantly reversed joint swelling, disability, grip strength impairment, and thermal and mechanical hypersensitivity. The effect of APHC3 was equal to or better than that of reference NSAIDs. Protracted treatment with APHC3 decreased IL-1b concentration in synovial fluid, reduced inflammatory changes in joints, and prevented the progression of cartilage degradation. Therefore, polypeptide APHC3 has the potential to be an analgesic and anti-inflammatory substance for the alleviation of arthritis symptoms.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Venenos de Cnidários/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Analgésicos/isolamento & purificação , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/fisiopatologia , Venenos de Cnidários/isolamento & purificação , Modelos Animais de Doenças , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Masculino , Osteoartrite/tratamento farmacológico , Osteoartrite/fisiopatologia , Dor/tratamento farmacológico , Dor/fisiopatologia , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
Assuntos
Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Sistema Livre de Células , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismoRESUMO
Acid-sensing ion channel 3 (ASIC3) makes an important contribution to the development and maintenance of inflammatory and acid-induced pain. We compared different ASIC3 inhibitors (peptides from sea anemones (APETx2 and Ugr9-1) and nonpeptide molecules (sevanol and diclofenac)) in anti-inflammatory action and analgesic effects. All tested compounds had distinct effects on pH-induced ASIC3 current. APETx2 inhibited only transient current, whereas Ugr9-1 and sevanol decreased transient and sustained components of the current. The effect on mice was evaluated after administering an intramuscular injection in the acetic acid writhing pain model and the complete Freund's adjuvant-induced thermal hyperalgesia/inflammation test. The bell-shaped dependence of the analgesic effect was observed for APETx2 in the acetic acid-induced writhing test, as well as for sevanol and peptide Ugr9-1 in the thermal hyperalgesia test. This dependence could be evidence of the nonspecific action of compounds in high doses. Compounds reducing both components of ASIC3 current produced more significant pain relief than APETx2, which is an effective inhibitor of a transient current only. Therefore, the comparison of the efficacy of ASIC3 inhibitors revealed the importance of ASIC3-sustained currents' inhibition for promotion of acidosis-related pain relief.
Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Analgésicos/farmacologia , Produtos Biológicos/farmacologia , Hiperalgesia/tratamento farmacológico , Dor/tratamento farmacológico , Anêmonas-do-Mar , Ácido Acético/toxicidade , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Diclofenaco/farmacologia , Modelos Animais de Doenças , Humanos , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Nociceptividade/efeitos dos fármacos , Dor/induzido quimicamente , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Xenopus laevisRESUMO
Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Ascomicetos/química , Anti-Infecciosos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Ascomicetos/metabolismo , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fungos/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Estrutura MolecularRESUMO
APHC3 is an analgesic polypeptide that was found in the sea anemone (Heteractis crispa), and contains 56 amino acid residues. This polypeptide is of interest for the development of medications for diseases, associated with inflammatory or neuropathological processes, as well as its use as an analgesic. This work presents an innovative biotechnological method for APHC3 production. We have constructed a recombinant plasmid intended for biosynthesizing the fusion protein consisting of a chitin-binding domain, DnaB mini-intein from Synechocystis sp. capable of undergoing pH-dependent self-cleavage, and the target peptide. In the process of biosynthesis the fusion protein aggregates and forms the inclusion bodies that are welcomed since APHC3 is a cytotoxic peptide. The target peptide recovery process developed by us involves 3 chromatographic steps. The method developed by us enables to produce 940â¯mg of the recombinant APHC3 from 100â¯g of the inclusion bodies. The method is straightforward to implement and scale up. The recombinant APHC3 activity and effectiveness as an analgesic was proved by animal testing.
Assuntos
Cromatografia/métodos , Venenos de Cnidários/isolamento & purificação , Expressão Gênica , Inteínas , Peptídeos/isolamento & purificação , Anêmonas-do-Mar/metabolismo , Animais , Clonagem Molecular , Venenos de Cnidários/genética , Escherichia coli/genética , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.
Assuntos
Venenos de Cnidários/química , Peptídeos/isolamento & purificação , Anêmonas-do-Mar , Animais , Venenos de Cnidários/genética , Peptídeos/análise , Anêmonas-do-Mar/química , Anêmonas-do-Mar/genética , Alinhamento de SequênciaRESUMO
TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone Heteractis crispa (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca2+ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3-300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 µM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength-potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.
Assuntos
Capsaicina/farmacologia , Canais de Cátion TRPV/metabolismo , Animais , Células CHO , Venenos de Cnidários/farmacologia , Cricetulus , Ligantes , Modelos Moleculares , Peptídeos/farmacologia , RatosRESUMO
A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.
Assuntos
Analgésicos , Antibacterianos , Anti-Inflamatórios , Peptídeos , Anêmonas-do-Mar , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Analgésicos/química , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dissulfetos/química , Edema/tratamento farmacológico , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/uso terapêuticoRESUMO
The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.
Assuntos
Analgésicos/farmacologia , Peptídeos/farmacologia , Anêmonas-do-Mar/química , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetulus , Camundongos , Peptídeos/química , Peptídeos/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 µg/g in experiments on house fly Musca domestica larvae and with LD50 20 µg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended ß-hairpin loop and short ß-hairpin loop which are capable of making "scissors-like mutual motions".
Assuntos
Bloqueadores dos Canais de Cálcio/toxicidade , Canais de Cálcio/metabolismo , Proteínas de Insetos/toxicidade , Venenos de Aranha/química , Aranhas/química , Sequência de Aminoácidos , Animais , Anuros , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/química , Células Cultivadas , Clonagem Molecular , Baratas/efeitos dos fármacos , Baratas/fisiologia , Dípteros/efeitos dos fármacos , Dípteros/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Alinhamento de Sequência , Venenos de Aranha/biossíntese , Aranhas/fisiologiaRESUMO
Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C(1)X3C(2)XnC(3)X3C(4)), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C(1)-C(4) and C(2)-C(3). Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal "tail" regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S-S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.
Assuntos
Antifúngicos/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Stellaria/química , Sequência de Aminoácidos , Antifúngicos/química , Clonagem Molecular , Evolução Molecular , Fungos/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sementes/química , Sementes/genética , Stellaria/metabolismoRESUMO
Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01-0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3's ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.