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1.
Iran Biomed J ; 27(4): 158-66, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37553755

RESUMO

Background: The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Methods: Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. Results: The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. Conclusion: This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.


Assuntos
Inseticidas , Venenos de Escorpião , Animais , Sequência de Aminoácidos , Escorpiões/química , Inseticidas/metabolismo , Venenos de Escorpião/genética , Cisteína/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo
2.
J Ethnopharmacol ; 265: 113268, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32810618

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: As well-known medicinal materials in traditional Chinese medicine, scorpions, commonly called as Quanxie () in Chinese, have been widely used to treat several diseases such as rheumatoid arthritis, apoplexy, epilepsy and chronic pain for more than a thousand years. Not only in the ancient times, the scorpions have also been recorded nowadays in the Pharmacopoeia of the People's Republic of China since 1963. AIM OF STUDY: This study aims to explore the differences in composition of the venom of scorpions from different regions by using the method of transcriptomics and proteomics. MATERIALS AND METHODS: Whole de novo transcriptomes, proteomics and their bioinformatic analyses were performed on samples of the scorpion Mesobuthus martensii and their venoms from four different provinces with clear geographical boundaries, including Hebei, Henan, Shandong and Shanxi. RESULTS: The four captured samples had the same morphology, and the conserved CO-1 sequence matched that of M. martensii. A total of 141,003 of 174,653 transcripts were identified as unigenes, of which we successfully annotated 51,627 (36.61%), 21,970 (15.58%), 7,168 (5.08%), and 45,263 (32.10%) unigenes with the NR, GO, KEGG and SWISSPROT databases, respectively, while a total of 427 proteins were collected from the protein extracted from venoms. Both GO and KEGG annotations exhibited only slight differences among the four samples while the expression level of gene and protein was quite different. A total of 249 toxin-related unigenes were successfully screened, including 41 serine proteases and serine protease inhibitors, 39 potassium channel toxins, 38 phospholipases, 16 host defense peptides, 9 metalloproteases, and 50 other toxins. Although the toxin species were similar among the four samples, the gene expression of each toxin varied considerably, for example, the scorpion from HB province has the most abundant expression quality in sequences c48391_g1, c55239_g1 and c47749_g1 while the lowest expressions of c51178_g1, c62033_g3 and c63754_g2. CONCLUSION: The regional differences in the transcriptomes and proteomes of M. martensii are mainly from expression levels e.g. toxins rather than expression species, of which the method can be further extended to evaluate the qualities of traditional Chinese medicines obtained from different regions.


Assuntos
Proteômica , Venenos de Escorpião/toxicidade , Escorpiões , Transcriptoma , Animais , China , Biologia Computacional , Expressão Gênica , Perfilação da Expressão Gênica , Proteoma , Venenos de Escorpião/química , Venenos de Escorpião/genética
3.
Toxicon ; 143: 1-19, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29305080

RESUMO

Scorpion, as an ancient species, has been widely used on dozens of human diseases in traditional Chinese Medicine. Although the scorpion venom from the Buthidae family with the potent toxicity attracts more interests, toxins from the non-Buthidae family draw great attention as well because of its abundance and complexity even without harm to mammals. Moreover, several toxic components of scorpion venom have been identified as valuable scaffolds for the drug design and development. Using the Next Generation Sequencing (NGS) technique, here we reported the transcriptome of the venomous glands of Heterometrus spinifer, a non-Buthidae scorpion that only a few toxic and complete components have been identified known-to-date. The total mRNA extracted from the venomous glands of H. spinifer was subjected to illumina sequencing with a strategy of de novo assembly, and a total of 54 189 transcripts were unigenes from a total of 88 311 600 determined reads. We annotated 18 567 (34.26%) unigenes from NR database, 12 258 (22.62%) from SWISSPROT database, 11 161 (20.60%) from GO database, 10 159 (18.75%) from COG database and 5059 (9.34%) from KEGG database, respectively. 2843 unigenes were further selected against the toxin-related sub-database of SWISSPROT. After removing the redundancy, 13 common toxin-related subfamilies with 62 unigenes were manually confirmed, including 8 K-toxins, 1 calcin, 3 Imperatoxin I-like, 2 La1-like, 1 scorpin-like, 3 antimicrobial peptides, two types of protease inhibitors such as 8 Kunitz-type protease inhibitors and 3 Ascaris-type protease inhibitors, and 33 proteases including 16 serine proteinases, 7 phospholipases, 5 metalloproteases, 3 hyaluronidases and 2 phosphatases. Our report is the first transcriptomic analyses of venomous glands from the scorpion H. spinifer, serving as a public information platform for the development of novel bio-therapeutics.


Assuntos
Proteínas de Artrópodes/análise , Perfilação da Expressão Gênica , Venenos de Escorpião/química , Escorpiões/metabolismo , Animais , Proteínas de Artrópodes/metabolismo , Glândulas Exócrinas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro , Venenos de Escorpião/genética , Escorpiões/genética
4.
Toxins (Basel) ; 8(12)2016 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-27941686

RESUMO

Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.


Assuntos
Proteínas de Artrópodes , Venenos de Escorpião , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Glândulas Exócrinas/metabolismo , Perfilação da Expressão Gênica , Filogenia , Proteômica , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Escorpiões
5.
Chin J Nat Med ; 14(8): 607-14, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27608950

RESUMO

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.


Assuntos
Inibidores de Proteases/química , Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Inibidores de Proteases/toxicidade , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Escorpiões/genética , Tripsina/química
6.
Insect Sci ; 23(2): 265-76, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25641865

RESUMO

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.


Assuntos
Antibiose , Arabidopsis/fisiologia , Herbivoria/efeitos dos fármacos , Insetos/fisiologia , Nicotiana/fisiologia , Oryza/fisiologia , Venenos de Escorpião/farmacologia , Animais , Arabidopsis/genética , Galanthus/química , Hemípteros/crescimento & desenvolvimento , Hemípteros/fisiologia , Insetos/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/fisiologia , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/farmacologia , Mariposas/crescimento & desenvolvimento , Mariposas/fisiologia , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Oryza/genética , Lectinas de Plantas/genética , Lectinas de Plantas/farmacologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Venenos de Escorpião/genética , Escorpiões/química , Nicotiana/genética
7.
J Sci Food Agric ; 94(6): 1163-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24027136

RESUMO

BACKGROUND: Various studies have been conducted to evaluate the effect of Bt crops on animals. Insect-resistant bioassays have revealed that the chitinase-BmkIT combination could be used as a new pest-resistant gene source and might be a complementary alien gene source to the Bt toxin gene. So it is necessary to assay the effects of chitinase-BmkIT plants on animals. RESULTS: Forty 40-day old New Zealand White Rabbits (Oryctolagus cuniculus) were fed for 60 consecutive days with formulated feed containing dehydrated poplar (Populus cathayana Rehd) leaves harbouring chitinase-BmkIT gene combination or untransformed counterparts, and the potential toxicological effects of transgenic leaves on rabbits were explored. The results of the growth study revealed no significant differences for daily weight gain, feed intake and feed conversion ratio where they were 101.6%, 99.2% and 97.8% of the treatment compared to the control, respectively. No obvious pathological change was observed in the small intestine, stomach, spleen, kidney, lung, heart, bladder, pancreas, prostate and ovary. Electron microscopy observations of liver cells and renal cells showed they were both normal in the two groups. No feed-derived chitinase, BmkIT and NPTII genes were found in small intestine, blood, or leg muscle samples although they were detected in the formulated feed. CONCLUSION: We conclude that the processed poplar leaves with foreign chitinase-BmkIT genes had no obviously harmful effects on rabbits.


Assuntos
Ração Animal , Dieta , Alimentos Geneticamente Modificados/efeitos adversos , Insetos/genética , Folhas de Planta , Plantas Geneticamente Modificadas , Populus/genética , Animais , Quitinases/genética , Ingestão de Alimentos , Metabolismo Energético , Feminino , Masculino , Coelhos , Venenos de Escorpião/genética , Distribuição Tecidual , Aumento de Peso
9.
Anal Bioanal Chem ; 405(7): 2379-89, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23307127

RESUMO

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Canal de Potássio Kv1.3/química , Microscopia Confocal/métodos , Animais , Escherichia coli/química , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Ligantes , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Escorpião/química , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Escorpiões , Esferoplastos/química , Esferoplastos/genética , Esferoplastos/metabolismo , Venenos de Aranha/química , Aranhas
10.
Biochem Biophys Res Commun ; 430(1): 113-8, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23200836

RESUMO

Potassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30-40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the ß-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T.stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the ß-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538 bp codifying a mature protein with 47 amino acid residues, corresponding to 5299 Da. This ß-KTx peptide is a new member of the BmTXKß-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade.


Assuntos
Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Modelos Químicos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptídeos/genética , Conformação Proteica , Venenos de Escorpião/genética , Escorpiões/genética
11.
Toxicon ; 61: 62-71, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23142507

RESUMO

Heteromtoxin (HmTx) is a group III phospholipase A(2) produced in Heterometrus laoticus, in Thailand. In this study, HmTx was purified from venom by separation chromatography, and the PLA(2) activity of the fractions was determined by lecithin agar assay. The enzyme is an acidic protein with a pI of 5.6 and an apparent molecular weight of 14018.4 Da. The nucleotide sequence of HmTx contains 649 bp, and the mature protein is predicted to have 131 amino acid residues-104 of which make up the large subunit, and 27 of which make up the small subunit. The subunit structure of HmTx is highly similar to that of the other toxin, Pandinus imperator imperatoxin I (IpTx(i)) and to Mesobuthus tamulus phospholipase A(2) (MtPLA(2)). The 3D-structure of HmTx consists of three conserved alpha-helices: h1 (Lys24-His34), h2 (Cys59-Asp71), and h3 (Ala80-Phe89). The beta-sheet consisted of a single stranded anti-parallel beta-sheet (b1.1 at Glu43-Lys45 and b1.2 at Lys48-Asn50) that was highly similar to the conserved sequences (-CGXG-, -CCXXHDXC- and CXCEXXXXXC-) of Apis mellifera (bee) phospholipases.


Assuntos
Fosfolipases A2/química , Venenos de Escorpião/enzimologia , Escorpiões/química , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A2/genética , Filogenia , Venenos de Escorpião/genética , Escorpiões/genética , Homologia de Sequência de Aminoácidos
12.
Proteomics ; 12(2): 313-28, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22121013

RESUMO

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7-30 µg and a paralysis or lethal effect was observed at 30 µg of venom (death at 1.5 µg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 µg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage-gated Na(+) and K(+) channels. Some fractions block K(+) currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC-MS/MS and MALDI-TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.


Assuntos
Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Estruturas Animais/química , Animais , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Dissulfetos/química , Estimulação Elétrica , Fenômenos Eletrofisiológicos , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Biblioteca Gênica , Gryllidae/efeitos dos fármacos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Mapeamento de Peptídeos/métodos , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/toxicidade , Canais de Potássio/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Escorpiões/genética , Canais de Sódio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Xenopus laevis/metabolismo
13.
PLoS One ; 6(8): e23520, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887265

RESUMO

BACKGROUND: Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na(+) channel isoforms. PRINCIPAL FINDINGS: C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Na(v)1.7, a Na(+) channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Na(v)1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Na(v)1.2, Na(v)1.3 and Na(v)1.4. The effects of CvIV4 are similar to Old World α-toxins that target Na(v)1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Na(v)1.7 channels (D1586A and E1589Q, DIV S3-S4 linker) reduced but did not abolish the effects of CvIV4. CONCLUSIONS: This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na(+) channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Na(v)1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.


Assuntos
Dor/patologia , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/toxicidade , Escorpiões/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Estruturas Animais/metabolismo , Animais , Sequência de Bases , Comportamento Animal/efeitos dos fármacos , Fracionamento Químico , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Dor/induzido quimicamente , Peptídeos/química , Peptídeos/isolamento & purificação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Venenos de Escorpião/química , Venenos de Escorpião/genética , Análise de Sequência de Proteína , Canais de Sódio/química , Canais de Sódio/metabolismo
14.
Biochimie ; 92(12): 1847-53, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20713119

RESUMO

Scorpion venom-derived peptidyl toxins are valuable pharmacological tools for investigating the structure-function relationship of ion channels. Here, we report the purification, sequencing and functional characterization of a new K(+) channel blocker (MeuKTX) from the venom of the scorpion Mesobuthus eupeus. Effects of MeuKTX on ten cloned potassium channels in Xenopus oocytes were evaluated using two-electrode voltage-clamp recordings. MeuKTX is the orthologue of BmKTX (α-KTx3.6), a known Kv1.3 blocker from the scorpion Mesobuthus martensii, and classified as α-KTx3.13. MeuKTX potently blocks rKv1.1, rKv1.2 and hKv1.3 channels with 50% inhibitory concentration (IC(50)) of 203.15 ± 4.06 pM, 8.92 ± 2.3 nM and 171 ± 8.56 pM, respectively, but does not affect rKv1.4, rKv1.5, hKv3.1, rKv4.3, and hERG channels even at 2 µM concentration. At this high concentration, MeuKTX is also active on rKv1.6 and Shaker IR. Our results also demonstrate that MeuKTX and BmKTX have the same channel spectrum and similar pharmacological potency. Analysis of the structure-function relationships of α-KTx3 subfamily toxins allows us to recognize several key sites which may be useful for designing toxins with improved activity on hKv1.3, an attractive target for T-cell mediated autoimmune diseases.


Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/farmacologia , Escorpiões/química , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/fisiologia , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp , Filogenia , Bloqueadores dos Canais de Potássio/classificação , Bloqueadores dos Canais de Potássio/isolamento & purificação , RNA Complementar/administração & dosagem , RNA Complementar/genética , Ratos , Venenos de Escorpião/genética , Venenos de Escorpião/isolamento & purificação , Homologia de Sequência de Aminoácidos , Xenopus laevis
15.
Mol Cell Biochem ; 330(1-2): 97-104, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19370317

RESUMO

The Asian scorpion Buthus martensil Karsch is important in the Chinese traditional medicine where it is used for the treatment of some nervous system diseases. The anti-epilepsy peptide (AEP) is a 61-amino-acid polypeptide extracted from the venom of B. martensil Karsch. Research has confirmed that it has anti-epileptic effects on the rat model of epilepsy. In this experiment, a cDNA library of AEP from the venom of B. martensil Karsch was constructed using RT-PCR; the primer was designed and used for the amplification. An expression vector of AEP was constructed using Pichia pastoris. Vector expression was induced, and protein purification was then performed. Bolting of the interaction molecule of AEP was by His pull down. Experimental results indicate high AEP expression, and the obtained protein was purified and compared with the control group. Four conspicuous protein bands were observed, and mass chromatographic analysis indicated that the four proteins were synaptosomal-associated protein of 25 kDa (SNAP-25), glial fibrillary acidic protein (GFAP), Glutamic acid decarboxylase (GAD) and N-methyl-D: -aspartate (NMDA). Further, the four protein bands were verified by mammalian two-hybrid experiments and co-immunoprecipitation. AEP was found to interact with SNAP2 and NMDA. This provides experimental evidence for the mechanism of AEP's anti-epileptic action and for the manufacture of a novel type anti-epileptic drug.


Assuntos
Clonagem Molecular/métodos , Epilepsia/tratamento farmacológico , Venenos de Escorpião/farmacologia , Animais , Vetores Genéticos , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Hipocampo/química , Hipocampo/citologia , N-Metilaspartato/metabolismo , Pichia/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Sprague-Dawley , Venenos de Escorpião/genética , Proteína 25 Associada a Sinaptossoma/metabolismo
16.
Proc Natl Acad Sci U S A ; 105(8): 3140-4, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18287010

RESUMO

Acid-sensing ion channels (ASICs) are neuronal non-voltage-gated cation channels that are activated when extracellular pH falls. They contribute to sensory function and nociception in the peripheral nervous system, and in the brain they contribute to synaptic plasticity and fear responses. Some of the physiologic consequences of disrupting ASIC genes in mice suggested that ASIC channels might modulate neuronal function by mechanisms in addition to their H(+)-evoked opening. Within ASIC channel's large extracellular domain, we identified sequence resembling that in scorpion toxins that inhibit K(+) channels. Therefore, we tested the hypothesis that ASIC channels might inhibit K(+) channel function by coexpressing ASIC1a and the high-conductance Ca(2+)- and voltage-activated K(+) (BK) channel. We found that ASIC1a associated with BK channels and inhibited their current. Reducing extracellular pH disrupted the association and relieved the inhibition. BK channels, in turn, altered the kinetics of ASIC1a current. In addition to BK, ASIC1a inhibited voltage-gated Kv1.3 channels. Other ASIC channels also inhibited BK, although acidosis-dependent relief of inhibition varied. These results reveal a mechanism of ion channel interaction and reciprocal regulation. Finding that a reduced pH activated ASIC1a and relieved BK inhibition suggests that extracellular protons may enhance the activity of channels with opposing effects on membrane voltage. The wide and varied expression patterns of ASICs, BK, and related K(+) channels suggest broad opportunities for this signaling system to alter neuronal function.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Canais de Sódio/genética , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Sequência de Aminoácidos , Linhagem Celular , DNA Complementar/genética , Eletrofisiologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Dados de Sequência Molecular , Neurônios/metabolismo , Estrutura Terciária de Proteína , Venenos de Escorpião/genética
17.
Peptides ; 28(12): 2306-12, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18006119

RESUMO

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.


Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Escorpiões/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Eletrofisiologia , Humanos , Canal de Potássio Kv1.3/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Técnicas de Patch-Clamp , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Peptides ; 28(1): 31-7, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17141373

RESUMO

Scorpine and toxins specific for potassium channels of the family beta (beta-Ktx) are two types of structurally related scorpion venom components, characterized by an unusually long extended N-terminal segment, followed by a Cys-rich domain with some resemblance to other scorpion toxins. In this communication, we report evidence supporting the ubiquitous presence of Scorpine and beta-KTx-like polypeptides and their precursors in scorpions of the genus Tityus of the family Buthidae, but also included is the first example of such peptides in scorpions from the family Iuridae. Seven new beta-KTxs or Scorpine-like peptides and precursors are reported: five from the genus Tityus (T. costatus, T. discrepans and T. trivittatus) and two from Hadrurus gertschi. The cDNA precursors for all of these peptides were obtained by molecular cloning and their presence in the venoms were confirmed for various peptides. Analysis of the sequences revealed the existence of at least three distinct groups: (1) beta-KTx-like peptides from buthids; (2) Scorpine-like peptides from scorpionid and iurid scorpions; (3) heterogeneous peptides similar to BmTXKbeta of buthids and iurids. The biological function for most of these peptides is not well known; that is why they are here considered "orphan" peptides.


Assuntos
Filogenia , Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Venenos de Escorpião/genética , Venenos de Escorpião/isolamento & purificação , Escorpiões/genética , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
19.
FEBS Lett ; 580(30): 6825-36, 2006 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-17141763

RESUMO

Venoms from scorpions contain extremely rich bioactive peptides that often carry diverse functions and are presumably needed to achieve synergistic effects for rapidly immobilizing prey and defending themselves. BotLVP1 is a unique heterodimer protein recently found in the scorpion Buthus occitanus tunetanus venom that is structurally related to scorpion toxins affecting sodium channels (NaScTxs) but exhibits adipocyte lipolysis activity. We have isolated and identified two cDNA clones encoding subunits alpha and beta of a BotLVP1-like peptide (named BmLVP1) from the Chinese scorpion Buthus martensii venom gland and determined the first complete gene structure of this subfamily. These results highlight a genetic link between these lipolysis activating peptides and NaScTxs. Comparison of cDNA and genomic sequences combined with protein structural and functional analysis provides evidence supporting the existence of RNA editing mechanism in scorpion venom glands, which could mediate functional switch of BmLVP1 gene, from adipocyte lipolysis to neurotoxicity, by altering the wrapper disulfide bridge (WDB) pattern of the peptides.


Assuntos
Dissulfetos/metabolismo , Lipólise , Peptídeos/metabolismo , Venenos de Escorpião/metabolismo , Escorpiões/metabolismo , Adenina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Estrutura Terciária de Proteína , Edição de RNA/genética , Venenos de Escorpião/química , Venenos de Escorpião/genética , Escorpiões/química , Escorpiões/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Canais de Sódio/genética , Canais de Sódio/metabolismo , Homologia Estrutural de Proteína , Transcrição Gênica/genética
20.
Peptides ; 26(5): 731-6, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15808902

RESUMO

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.


Assuntos
Neurotoxinas/genética , Peptídeos/genética , Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Proteínas de Insetos , Dados de Sequência Molecular , Neurotoxinas/química , Peptídeos/química , Venenos de Escorpião/química , Escorpiões/metabolismo , Análise de Sequência de DNA , Análise de Sequência de Proteína
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