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1.
Biochem J ; 477(6): 1149-1158, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32150261

RESUMO

Searching for compounds that inhibit the growth of photosynthetic organisms highlighted a prominent effect at micromolar concentrations of the nitroheteroaromatic thioether, 2-nitrothiophene, applied in the light. Since similar effects were reminiscent to those obtained also by radicals produced under excessive illumination or by herbicides, and in light of its redox potential, we suspected that 2-nitrothiophene was reduced by ferredoxin, a major reducing compound in the light. In silico examination using docking and tunneling computing algorithms of the putative interaction between 2-nitrothiophene and cyanobacterial ferredoxin has suggested a site of interaction enabling robust electron transfer from the iron-sulfur cluster of ferredoxin to the nitro group of 2-nitrothiophene. ESR and oximetry analyses of cyanobacterial cells (Anabaena PCC7120) treated with 50 µM 2-nitrothiophene under illumination revealed accumulation of oxygen radicals and peroxides. Gas chromatography mass spectrometry analysis of 2-nitrothiophene-treated cells identified cytotoxic nitroso and non-toxic amino derivatives. These products of the degradation pathway of 2-nitrohiophene, which initializes with a single electron transfer that forms a short-live anion radical, are then decomposed to nitrate and thiophene, and may be further reduced to a nitroso hydroxylamine and amino derivatives. This mechanism of toxicity is similar to that of nitroimidazoles (e.g. ornidazole and metronidazole) reduced by ferredoxin in anaerobic bacteria and protozoa, but differs from that of ornidazole in planta.

2.
Proc Natl Acad Sci U S A ; 116(37): 18700-18709, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31444298

RESUMO

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

4.
Biochem J ; 473(23): 4413-4426, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27647935

RESUMO

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP+ reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle.


Assuntos
Bactérias Anaeróbias/efeitos dos fármacos , Ornidazol/farmacologia , Fotossíntese/efeitos dos fármacos , Cianobactérias/efeitos dos fármacos , Ferredoxinas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Metronidazol/farmacologia , Modelos Biológicos , Synechocystis/efeitos dos fármacos , Trichomonas/efeitos dos fármacos , Trichomonas/metabolismo , Triose-Fosfato Isomerase/metabolismo
5.
Biochem J ; 463(2): 271-7, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25055135

RESUMO

Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site-3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3 was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determining Av3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp404 and His405 as a putative point of Av3 interaction with DmNav1. His405 conservation in arthropod Navs compared with tyrosine in vertebrate Navs may represent an ancient substitution that explains the contemporary selectivity of Av3. Trp404 and His405 localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/química , Drosophila/metabolismo , Toxinas Marinhas/química , Anêmonas-do-Mar/metabolismo , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/química , Canais de Sódio/metabolismo , Animais , Sítios de Ligação , Drosophila/efeitos dos fármacos , Drosophila/genética , Proteínas de Drosophila/genética , Toxinas Marinhas/metabolismo , Toxinas Marinhas/toxicidade , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Anêmonas-do-Mar/química , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/genética , Xenopus laevis
6.
PLoS One ; 8(11): e77758, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24302985

RESUMO

The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 (+) trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.


Assuntos
Venenos de Escorpião/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Linhagem Celular , Humanos , Cinética , Mesilatos/metabolismo , Mesilatos/farmacologia , Ligação Proteica , Venenos de Escorpião/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/química
7.
Cell Rep ; 2(2): 242-8, 2012 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-22854023

RESUMO

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.


Assuntos
Cálcio/metabolismo , Evolução Molecular , Anêmonas-do-Mar , Sódio/metabolismo , Transmissão Sináptica/fisiologia , Canais de Sódio Disparados por Voltagem , Sequência de Aminoácidos , Animais , Transporte de Íons/fisiologia , Dados de Sequência Molecular , Neurônios/metabolismo , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo
8.
J Biol Chem ; 287(36): 30719-28, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22761417

RESUMO

Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. ß-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for ß-scorpion toxins on Na(V) channels. Binding of α- and ß-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Venenos de Escorpião/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Ativação do Canal Iônico/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos
9.
Toxicon ; 60(4): 502-11, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22694883

RESUMO

Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain II, while Lqh2 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa(v)1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module.


Assuntos
Neurotoxinas/metabolismo , Receptores de Superfície Celular/metabolismo , Venenos de Escorpião/metabolismo , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ativação do Canal Iônico , Dados de Sequência Molecular , Neurotoxinas/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Venenos de Escorpião/química , Alinhamento de Sequência , Canais de Sódio/química , Especificidade da Espécie , Relação Estrutura-Atividade
10.
Proc Biol Sci ; 279(1732): 1351-8, 2012 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-22048953

RESUMO

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.


Assuntos
Venenos de Cnidários/metabolismo , Neurotoxinas/metabolismo , Anêmonas-do-Mar/fisiologia , Animais , Artemia , Evolução Biológica , Venenos de Cnidários/genética , Venenos de Cnidários/toxicidade , Imuno-Histoquímica , Neurotoxinas/genética , Neurotoxinas/toxicidade , Comportamento Predatório , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Anêmonas-do-Mar/anatomia & histologia , Anêmonas-do-Mar/genética , Peixe-Zebra
11.
J Biol Chem ; 286(40): 35209-17, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21832067

RESUMO

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.


Assuntos
Venenos de Escorpião/metabolismo , Escorpiões/metabolismo , Canais de Sódio/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , DNA Complementar/metabolismo , Drosophila , Conformação Molecular , Dados de Sequência Molecular , Mutagênese , Mutação , Neurotoxinas/metabolismo , Ratos , Anêmonas-do-Mar , Homologia de Sequência de Aminoácidos , Xenopus
12.
Proc Natl Acad Sci U S A ; 108(37): 15426-31, 2011 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-21876146

RESUMO

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.


Assuntos
Venenos de Escorpião/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Aminoácidos/metabolismo , Ativação do Canal Iônico , Cinética , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo
13.
J Biol Chem ; 286(38): 33641-51, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21795675

RESUMO

Voltage-gated sodium (Na(v)) channels are the molecular targets of ß-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of ß-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Animais , Ativação do Canal Iônico , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-Atividade
14.
J Exp Bot ; 62(12): 4173-82, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21551078

RESUMO

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.


Assuntos
Mutagênese , Fotossíntese/genética , Ribulose-Bifosfato Carboxilase/genética , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Substituição de Aminoácidos/genética , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Clorofila/metabolismo , Ativação Enzimática/efeitos dos fármacos , Cinética , Mutagênese/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Oxigênio/metabolismo , Fosfatos/farmacologia , Fotossíntese/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/genética , Synechocystis/ultraestrutura
15.
J Biol Chem ; 286(18): 15781-8, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21454658

RESUMO

Scorpion ß-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant ß-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion ß-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion ß-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.


Assuntos
Blattellidae/metabolismo , Proteínas de Insetos/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Canais de Sódio/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Processamento Alternativo/fisiologia , Substituição de Aminoácidos , Animais , Blattellidae/química , Blattellidae/genética , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Venenos de Escorpião/química , Escorpiões/química , Canais de Sódio/química , Canais de Sódio/genética , Xenopus
16.
J Biol Chem ; 285(40): 30531-8, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20682774

RESUMO

Scorpion ß-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.


Assuntos
Substituição de Aminoácidos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Animais , Sítios de Ligação , Mordeduras e Picadas/terapia , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Camundongos , Mutação , Ratos , Ratos Wistar , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/uso terapêutico , Escorpiões
17.
Biophys J ; 99(2): 456-63, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20643063

RESUMO

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.


Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Canais de Sódio/química , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Estrutura Secundária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade
18.
Mol Biol Evol ; 27(5): 1025-34, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20018978

RESUMO

Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.


Assuntos
Aminoácidos/genética , Evolução Molecular , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Seleção Genética , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Insetos , Ativação do Canal Iônico/efeitos dos fármacos , Funções Verossimilhança , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacologia , Filogenia , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Escorpiões/classificação , Escorpiões/genética , Canais de Sódio/metabolismo
19.
J Mol Evol ; 69(2): 115-24, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19609479

RESUMO

Sea anemones are sessile predators that use a variety of toxins to paralyze prey and foe. Among these toxins, Types I, II and III are short peptides that affect voltage-gated sodium channels. Anemonia viridis is the only sea anemone species that produces both Types I and III neurotoxin. Although the two toxin types are unrelated in sequence and three-dimensional structure, cloning and comparative analysis of their loci revealed a highly similar sequence at the 5' region, which encodes a signal peptide. This similarity was likely generated by gene fusion and could be advantageous in transcript stability and intracellular trafficking and secretion. In addition, these analyses identified the processed pseudogenes of the two gene families in the genome of A. viridis, probably resulting from retrotransposition events. As presence of processed pseudogenes in the genome requires transcription in germ-line cells, we analyzed oocyte-rich ovaries and found that indeed they contain Types I and III transcripts. This result raises questions regarding the role of toxin transcripts in these tissues. Overall, the retrotransposition and gene fusion events suggest that the genes of both Types I and III neurotoxins evolved in a similar fashion and share a partial common ancestry.


Assuntos
Evolução Molecular , Fusão Gênica , Neurotoxinas/genética , Retroelementos/genética , Anêmonas-do-Mar/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , DNA Intergênico/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/metabolismo , Filogenia , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos
20.
J Biol Chem ; 284(35): 23558-63, 2009 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-19574227

RESUMO

Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSalphabeta scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion beta-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion beta-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhalphaIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa(v)1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa(v)1 activation. Thus, the short CSalphabeta scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa(v)1.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/farmacologia , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Fungos/efeitos dos fármacos , Imunidade Inata , Dados de Sequência Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação Proteica , Alinhamento de Sequência
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