RESUMEN
Tetrodotoxin-sensitive persistent sodium currents, INaP, that activate at subthreshold voltages, have been detected in numerous vertebrate and invertebrate neurons. These currents are believed to be critical for regulating neuronal excitability. However, the molecular mechanism underlying INaP is controversial. In this study, we identified an INaP with a broad range of voltage dependence, from -60mV to 20mV, in a Drosophila sodium channel variant expressed in Xenopus oocytes. Mutational analysis revealed that two variant-specific amino acid changes, I260T in the S4-S5 linker of domain I (ILS4-S5) and A1731V in the voltage sensor S4 of domain IV (IVS4), contribute to the INaP. I260T is critical for the portion of INaP at hyperpolarized potentials. The T260-mediated INaP is likely the result of window currents flowing in the voltage range where the activation and inactivation curves overlap. A1731V is responsible for impaired inactivation and contributes to the portion of INaP at depolarized potentials. Furthermore, A1731V causes enhanced activity of two site-3 toxins which induce persistent currents by inhibiting the outward movement of IVS4, suggesting that A1731V inhibits the outward movement of IVS4. These results provided molecular evidence for the involvement of distinct mechanisms in the generation of INaP: T260 contributes to INaP via enhancement of the window current, whereas V1731 impairs fast inactivation probably by inhibiting the outward movement of IVS4.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Potenciales de la Membrana/fisiología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Animales , Proteínas de Drosophila/química , Drosophila melanogaster , Estimulación Eléctrica , Técnicas de Transferencia de Gen , Variación Genética , Potenciales de la Membrana/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Neurotoxinas/farmacología , Técnicas de Placa-Clamp , Venenos de Escorpión/farmacología , Canales de Sodio/química , XenopusRESUMEN
The structure of bioactive surfaces of proteins is a subject of intensive research, yet the mechanisms by which such surfaces have evolved are largely unknown. Polypeptide toxins produced by venomous animals such as sea anemones, cone snails, scorpions, and snakes show multiple routes for active site diversification, each maintaining a typical conserved scaffold. Comparative analysis of an array of genetically related scorpion polypeptide toxins that modulate sodium channels in neuronal membranes suggests a unique route of toxic site diversification. This premise is based on recent identification of bioactive surfaces of toxin representative of three distinct pharmacological groups and a comparison of their 3-dimensional structures. Despite their similar scaffold, the bioactive surfaces of the various toxins vary considerably, but always coincide with the molecular exterior onto which the C-tail is anchored. Superposition of the toxin structures indicates that the C-tails diverge from a common structural start point, which suggests that the pharmacological versatility displayed by these toxins might have been achieved along evolution via structural reconfiguration of the C-tail, leading to reshaping of new bioactive surfaces.
Asunto(s)
Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Venenos de Escorpión/genética , Venenos de Escorpión/farmacología , Alineación de Secuencia , Canales de Sodio/metabolismoRESUMEN
The alpha-like toxin from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity to receptor site 3 on insect sodium channels but does not bind to rat brain synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase in the electropositive charge on the toxin surface resulting from protonation of its four histidines. Radioiodination of Tyr(14) of Lqh III abolished its binding to locust but not cockroach sodium channels, whereas the noniodinated toxin bound equally well to both neuronal preparations. Radioiodination of Tyr(10) or Tyr(21) of the structurally similar alpha-toxin from L. quinquestriatus hebraeus (LqhalphaIT), as well as their substitution by phenylalanine, had only minor effects on binding to cockroach neuronal membranes. However, substitution of Tyr(21), but not Tyr(14), by leucine decreased the binding affinity of LqhalphaIT approximately 87-fold. Thus, Tyr(14) is involved in the bioactivity of Lqh III to locust receptor site 3 and is not crucial for the binding of LqhalphaIT to this site. In turn, the aromatic ring of Tyr(21) takes part in the bioactivity of LqhalphaIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.
Asunto(s)
Yodo/química , Neurotoxinas/metabolismo , Venenos de Escorpión/metabolismo , Canales de Sodio/metabolismo , Sustitución de Aminoácidos/genética , Animales , Unión Competitiva/genética , Saltamontes , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neurotoxinas/farmacología , Periplaneta , Estructura Terciaria de Proteína , Venenos de Escorpión/química , Venenos de Escorpión/genética , Sinaptosomas/metabolismo , Tirosina/químicaRESUMEN
Orthophosphate (Pi) modulates the activity and activation of ribulose 1,5-bis-phosphate carboxylase/oxygenase (RuBisCO) via a mechanism that is still controversial. Whereas its effects on the higher plant enzyme have been described, little is known about Pi regulation of the structurally similar, yet kinetically different cyanobacterial enzyme. We found that RuBisCO of Synechocystis PCC6803 was affected by Pi in a paradoxical fashion. On the one hand, Pi inhibited catalysis by competing with the substrate RuBP, and on the other hand it stimulated enzyme activation in a dual manner manifested by multiphasic kinetics, which differed from the effect on activation of the higher plant enzyme. Pi concentrations > 5 mM promoted the carbamylation of the cyanobacterial enzyme and the binding of Mg2+ to the carbanion at suboptimal concentrations of CO2 and Mg2+. Surprisingly, Pi also increased the activation level of the carbamylated enzyme via another putative site of interaction. In contrast with the higher plant RuBisCO, RuBP did not inhibit the stimulatory effect of phosphate on activation of the cyanobacterial enzyme, suggesting a Pi effect through a site other than the sugar binding site. The dual effect on activation could be distinguished by the phosphate analogue vanadate, which inhibited only the stimulation achieved at high phosphate concentrations. The elevation of RuBisCO activation at suboptimal levels of CO2 and high concentrations of RuBP suggests that in cyanobacteria Pi may have a role analogous to that of RuBisCO activase in higher plants.
Asunto(s)
Cianobacterias/enzimología , Fosfatos/farmacología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Sitios de Unión , Dióxido de Carbono/metabolismo , Catálisis/efectos de los fármacos , Cianobacterias/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Cinética , Modelos Químicos , Pentosafosfatos/metabolismo , Proteínas de Plantas/metabolismo , Ribulosa-Bifosfato Carboxilasa/efectos de los fármacos , Especificidad de la Especie , Alcoholes del Azúcar/metabolismo , Vanadatos/farmacologíaRESUMEN
The peptide LqhalphaIT is an alpha-scorpion toxin that shows significant selectivity for insect sodium channels over mammalian channels. We examined the symptoms of LqhalphaIT-induced paralysis and its neurophysiological correlates in the house fly (Musca domestica). Injection of LqhalphaIT into fly larvae produced hyperactivity characterized by continuous, irregular muscle twitching throughout the body. These symptoms were correlated with elevated excitability in motor units caused by two physiological effects of the toxin: 1) increased transmitter release and 2) repetitive action potentials in motor nerves. Increased transmitter release was evident as augmentation of neurally evoked synaptic current, and this was correlated with an increased duration of action potential-associated current (APAC) in loose patch recordings from nerve terminals. Repetitive APACs were observed to invade nerve endings. The toxin produced marked inhibition of sodium current inactivation in fly central neurons, which can account for increased duration of the APAC and elevated neurotransmitter release at the neuromuscular junction. Steady-state inactivation was shifted significantly to more positive potentials, whereas voltage-dependent activation of the channels was not affected. The shift in steady-state inactivation provides a mechanism for inducing repetitive activity in motoneurons. The effects of LqhalphaIT on sodium channel inactivation in motor nerve endings can account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.
Asunto(s)
Moscas Domésticas/fisiología , Neurotoxinas/farmacología , Venenos de Escorpión/farmacología , Bloqueadores de los Canales de Sodio , Transmisión Sináptica/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Larva , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Actividad Motora/efectos de los fármacos , Parálisis/inducido químicamente , Técnicas de Placa-Clamp , Estimulación Química , Sinapsis/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.
Asunto(s)
Canales de Cloruro/efectos de los fármacos , Neurotoxinas/genética , Canales de Potasio/efectos de los fármacos , Venenos de Escorpión/química , Canales de Sodio/efectos de los fármacos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario , Intrones , Datos de Secuencia Molecular , Neurotoxinas/farmacología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.
Asunto(s)
Cucarachas/efectos de los fármacos , Dípteros/efectos de los fármacos , Neurotoxinas/metabolismo , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , Clonación Molecular , Cartilla de ADN , Proteínas de Insectos , Potenciales de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/genética , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The preservation along evolution of specific core motifs in proteins of diverse functions and taxonomic origins pinpoints a possible developmental advantage at the structural level. Such a preservation was observed in a group of membrane potential modulators including plant gamma-thionins, scorpion toxins, insect and scorpion defensins, bee venom apamin and MCD peptide, snake sarafotoxins, and human endothelins. These substances are short polypeptides of various lengths and nonhomologous sequences that affect organisms of distant phyla. Despite the structural differences, comparative analysis reveals commonality at three levels: 1) effect on membrane potential; 2) a common cysteine-stabilized alpha-helical (CSH) motif; and 3) similar gene organization (except for insect defensins), i.e., an intron that splits a codon toward the end of the leader sequence. We thus propose that these modulators, divided into two groups differing in their CSH motif orientation, have either diverged from two independent ancestors or have evolved by gene diversification via exon shuffling and subsequent modifications. To enforce protein synthesis through the secretory pathway and enable disulfide bond formation and secretion, insertion sites downstream of preexisting leader sequences have been a prerequisite. What seems advantageous for evolution, may also be exploited in attempts to 'accelerate evolution' by protein design using the conserved CSH core as a suitable scaffold for reshaping molecular exteriors.
Asunto(s)
Proteínas de Arabidopsis , Evolución Molecular , Potenciales de la Membrana/efectos de los fármacos , Neurotoxinas/farmacología , Péptidos/farmacología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos , Endotelinas/química , Endotelinas/farmacología , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/clasificación , Péptidos/química , Péptidos/clasificación , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Ponzoñas/química , Ponzoñas/farmacologíaRESUMEN
BACKGROUND: Scorpion neurotoxins, which bind and modulate sodium channels, have been divided into two groups, the alpha and beta toxins, according to their activities. The beta-toxin class includes the groups of excitatory and depressant toxins, which differ in their mode of action and are highly specific against insects. The three-dimensional structures of several alpha and beta toxins have been determined at high resolution, but no detailed 3D structure of an excitatory toxin has been presented so far. RESULTS: The crystal structure of an anti-insect excitatory toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined at 2.1 A resolution and refined to an R factor of 0.209. The first 59 residues form a closely packed module, structurally similar to the conserved alpha and beta toxins ('long toxins') affecting sodium channels. The last 17 residues form a C-terminal extension not previously seen in scorpion toxins. It comprises a short alpha helix anchored to the N-terminal module by a disulfide bridge and is followed by a highly mobile stretch of seven residues, of which only four are seen in the electron-density map. This mobile peptide covers part of a conserved hydrophobic surface that is thought to be essential for interaction with the channel in several long toxins. CONCLUSIONS: Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.
Asunto(s)
Neurotoxinas/química , Neurotoxinas/metabolismo , Estructura Secundaria de Proteína , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Procesamiento de Imagen Asistido por Computador , Proteínas de Insectos , Insectos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Venenos de Escorpión/química , Escorpiones , Alineación de Secuencia , Relación Estructura-Actividad , Propiedades de SuperficieAsunto(s)
Neurotoxinas/farmacología , Venenos de Escorpión/química , Bloqueadores de los Canales de Sodio , Animales , Sinergismo Farmacológico , Escherichia coli , Predicción , Insecticidas/farmacología , Modelos Moleculares , Neurotoxinas/química , Neurotoxinas/genética , Polimorfismo Genético , Venenos de Escorpión/genética , Venenos de Escorpión/farmacología , Spodoptera , Relación Estructura-ActividadRESUMEN
The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti-insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild-type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.
Asunto(s)
Insecticidas , Neurotoxinas/toxicidad , Venenos de Escorpión/toxicidad , Secuencia de Aminoácidos , Animales , Baculoviridae , Línea Celular , Insectos , Cinética , Datos de Secuencia Molecular , Parálisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidad , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/química , Spodoptera , TransfecciónRESUMEN
alpha-Neurotoxins from scorpion venoms constitute the most studied group of modifiers of the voltage-sensitive sodium channels, and yet, their toxic site has not been characterized. We used an efficient bacterial expression system for modifying specific amino acid residues of the highly insecticidal alpha-neurotoxin LqhalphaIT from the scorpion Leiurus quinquestriatus hebraeus. Toxin variants modified at tight turns, the C-terminal region, and other structurally related regions were subjected to neuropharmacological and structural analyses. This approach highlighted both aromatic (Tyr10 and Phe17) and positively charged (Lys8, Arg18, Lys62, and Arg64) residues that (i) may interact directly with putative recognition points at the receptor site on the sodium channel; (ii) are important for the spatial arrangement of the toxin polypeptide; and (iii) contribute to the formation of an electrostatic potential that may be involved in biorecognition of the receptor site. The latter was supported by a suppressor mutation (E15A) that restored a detrimental effect caused by a K8D substitution. The feasibility of producing anti-insect scorpion neurotoxins with augmented toxicity was demonstrated by the substitution of the C-terminal arginine with histidine. Altogether, the present study provides for the first time an insight into the putative toxic surface of a scorpion neurotoxin affecting sodium channel gating.
Asunto(s)
Neurotoxinas/química , Conformación Proteica , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Dípteros , Larva , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neurotoxinas/toxicidad , Mutación Puntual , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/toxicidad , Escorpiones , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.
Asunto(s)
Neurotoxinas/genética , Venenos de Escorpión/química , Animales , Axones/efectos de los fármacos , Bacteriófago T7/genética , Cromatografía Líquida de Alta Presión , Cucarachas/efectos de los fármacos , Dípteros/efectos de los fármacos , Dípteros/crecimiento & desarrollo , Estabilidad de Medicamentos , Escherichia coli , Genes de Insecto , Genes Virales , Activación del Canal Iónico/efectos de los fármacos , Larva , Mutagénesis Sitio-Dirigida , Neurotoxinas/aislamiento & purificación , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Parálisis/inducido químicamente , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Venenos de Escorpión/toxicidad , Solubilidad , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The solution structure of a recombinant active alpha-neurotoxin from Leiurus quinquestriatus hebraeus, Lqh(alpha)IT, was determined by proton two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion alpha-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 A. These structures had root mean square deviations of 0.49 and 1.00 A with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of Lqh(alpha)IT consists of an alpha-helix, a three-strand antiparallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a "herringbone" arrangement. Positive phi angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh(alpha)IT is very similar to that of the most active antimammalian scorpion alpha-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified Lqh(alpha)IT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of Lqh(alpha)IT.
Asunto(s)
Neurotoxinas/química , Neurotoxinas/genética , Proteínas Recombinantes/química , Venenos de Escorpión/química , Venenos de Escorpión/genética , Secuencia de Aminoácidos , Arginina/genética , Histidina/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neurotoxinas/toxicidad , Control Biológico de Vectores , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/toxicidad , Venenos de Escorpión/toxicidad , SolucionesRESUMEN
The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.
Asunto(s)
Potenciales de la Membrana/efectos de los fármacos , Venenos de Escorpión/farmacología , Animales , Cinética , Masculino , Periplaneta/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Venenos de Escorpión/químicaRESUMEN
The alpha neurotoxin Lqh alpha IT is toxic to both insects and mammals but exhibits a bioactivity ratio favoring insects (insect/mammal approximately 2). With the objective of increasing this ratio by genetic manipulation of the amino acid sequence, a cDNA clone encoding Lqh alpha IT was used to produce recombinant variants of the toxin in a high efficiency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemical and biological properties indistinguishable from those of the authentic native toxin. Alteration of the toxin by site-directed mutagenesis led to a substantial reduction in anti-mammalian toxicity (mouse LD50 reduced 6.4-fold) but only a slight reduction (x 1.5) in the insect ED50 value for paralysis. The reduction in anti-mammalian toxicity was correlated with a approximately 2-fold reduction of its potency for slowing of sodium channel inactivation in mammalian neurons, while no change in mutant toxin binding affinity to insect neuronal receptors was registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and the use of site-directed mutagenesis to improve phylogenetic selectivity. This recombinant approach provides a promising strategy for optimizing the selective toxicity of peptide neurotoxins.
Asunto(s)
Neuronas/fisiología , Neurotoxinas/toxicidad , Venenos de Escorpión/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/ultraestructura , Ganglios Espinales/fisiología , Insectos , Isopropil Tiogalactósido , Mamíferos , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas/química , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidad , Venenos de Escorpión/química , Venenos de Escorpión/toxicidad , Escorpiones , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Canales de Sodio/efectos de los fármacos , Especificidad de la EspecieRESUMEN
The Leiurus quinquestriatus hebraeus alpha anti-insect toxin (Lqh alpha IT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional Lqh alpha IT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.
Asunto(s)
Neurotoxinas/genética , Venenos de Escorpión/genética , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Dípteros/efectos de los fármacos , Vectores Genéticos , Larva , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Neurotoxinas/toxicidad , Nucleopoliedrovirus/genética , Proteínas Recombinantes , Venenos de Escorpión/toxicidad , Spodoptera/efectos de los fármacosRESUMEN
The upstream noncoding region of the Synechococcus sp. strain PCC 7942 (hereafter referred to as Synechococcus 7942) glnA gene was fused to the cat gene in order to study the expression of glnA both in Synechococcus 7942 and in Escherichia coli. The lack of cat expression in E. coli indicated that the glnA promoter was not recognized by E. coli RNA polymerase. The fused construct was integrated into the Synechococcus 7942 chromosome at a neutral site. Expression of the cat reporter gene was regulated under various nitrogen conditions in a way similar to that of the glnA gene. A deletion introduced at the binding site of the NtcA regulatory protein abolished derepression of the glnA promoter during growth in nitrate and under nitrogen starvation. Deletion of the sequence between the transcription and translation start sites of glnA prevented the repression observed during growth in ammonium. These results indicate that the glnA promoter is subject to complex regulation that involves sequences upstream and downstream from the transcription start site.
Asunto(s)
Cianobacterias/genética , Genes Bacterianos , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
A high CO2-requiring mutant of Synechocystis PCC6803 (G3) capable of Ci transport but unable to utilize the intracellular Ci pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.
RESUMEN
The glnA mRNA, encoding glutamine synthetase, is differentially accumulated in the cyanobacterium Synechococcus sp. strain PCC 7942 in media containing different nitrogen sources. With the different nitrogen compounds, transcription of glnA initiated at a single site located -146 nucleotides upstream of the translation start site of the gene. A similarity of the nif-like promoter of the glnA gene of Anabaena sp. strain PCC 7120 and a binding-site sequence for the Synechococcus sp. strain PCC 7942 transcription regulator, NtcA, were found upstream of the transcription initiation site.