RESUMO
Introduction: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. Methods: In this study, we explored the neuroactivity and molecular diversity of P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Results: Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Discussion: Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggest that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.
RESUMO
Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.
Assuntos
Aranha Marrom Reclusa/genética , Miniproteínas Nó de Cistina/química , Inseticidas/análise , Diester Fosfórico Hidrolases/química , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Aranha Marrom Reclusa/metabolismo , Sequência Conservada , Miniproteínas Nó de Cistina/biossíntese , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/isolamento & purificação , Dípteros , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Dados de Sequência Molecular , Proteoma , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Testes de Toxicidade , TranscriptomaRESUMO
Histidine kinases are ubiquitous molecular sensors that are used by bacteria to detect and respond to a myriad of environmental signals. They are attractive antimicrobial targets because of their roles in mediating the virulence of pathogenic organisms, as well as the ability of bacteria to resist host defenses and develop resistance to antibiotics. In this review, we discuss the challenges involved in developing specific inhibitors of this highly diverse group of kinases.
Assuntos
Anti-Infecciosos/farmacologia , Sistemas de Liberação de Medicamentos , Proteínas Quinases/química , Proteínas Quinases/classificação , Animais , Anti-Infecciosos/química , Sistemas de Liberação de Medicamentos/tendências , Inibidores Enzimáticos/farmacologia , Histidina Quinase , Humanos , Proteínas Quinases/efeitos dos fármacosRESUMO
Limited proteolysis of the Escherichia coli cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain. We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Divisão Celular , Dimerização , Escherichia coli/citologia , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Prolina , Análise de Sequência de ProteínaRESUMO
We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega-agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Inseticidas/farmacologia , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Abelhas/citologia , Controle de Insetos/métodos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurotoxinas/genética , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Precursores de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Venenos de Aranha/química , Venenos de Aranha/genética , Aranhas , ômega-Agatoxina IVA/químicaRESUMO
omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.
Assuntos
Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Escherichia coli , Inseticidas/metabolismo , Inseticidas/toxicidade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Venenos de Aranha/metabolismo , Venenos de Aranha/toxicidade , Relação Estrutura-AtividadeRESUMO
Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular , Sequência Conservada , Dimerização , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.
Assuntos
Dissulfetos/química , Dissulfetos/metabolismo , Inseticidas/química , Inseticidas/isolamento & purificação , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Evolução Molecular , Insetos/citologia , Insetos/efeitos dos fármacos , Insetos/metabolismo , Inseticidas/toxicidade , Dose Letal Mediana , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Colinérgicos/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Venenos de Aranha/químicaRESUMO
delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.
Assuntos
Bloqueadores dos Canais de Sódio , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/farmacologia , Aranhas/química , Toxinas Biológicas/isolamento & purificação , Toxinas Biológicas/farmacologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Condutividade Elétrica , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gryllidae/efeitos dos fármacos , Inseticidas/química , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Alinhamento de Sequência , Análise de Sequência de Proteína , Sódio/metabolismo , Canais de Sódio/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Tetrodotoxina/farmacologia , Toxinas Biológicas/químicaRESUMO
We have isolated and determined the amino acid sequence of a novel peptide component from the venom of the Australian funnel-web spider Hadronyche versuta. This 68-residue toxin, ACTX-Hvf17, does not function like classical neurotoxins in modulating ion channel function as evidenced by its lack of insecticidal activity and its inability to affect vertebrate smooth or skeletal muscle contractility. The peptide shows significant sequence homology with mamba intestinal toxin 1 (MIT1) and to a lesser extent with a variety of colipases. The strong structural homology between MIT1 and porcine colipase leads us to propose that ACTX-Hvf17 also adopts the MIT1/colipase three-dimensional fold. However, we show that ACTX-Hvf17 has no colipase activity and does not stimulate muscle contractility like MITI. We also show that MIT1 and ACTX-Hvf17 display significant sequence homology with the C-terminal cysteine-rich domain of the Dickkopf-1 family of proteins that induce head formation in developing embryos, which leads us to propose that this domain of Dickkopf-1 also adopts the MIT1 colipase fold.
Assuntos
Peptídeos/química , Proteínas/química , Venenos de Aranha/química , Aranhas/metabolismo , Peçonhas/química , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Cisteína/análise , Dissulfetos/análise , Feminino , Liofilização , Gryllidae , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Oxirredução , Peptídeos/isolamento & purificação , Peptídeos/toxicidade , Proteínas/isolamento & purificação , Ratos , Homologia de Sequência de Aminoácidos , Venenos de Aranha/isolamento & purificação , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervação , Peçonhas/isolamento & purificaçãoRESUMO
The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Peptídeos/química , Venenos de Aranha/química , Tenebrio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Inseticidas/química , Inseticidas/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/farmacologia , Análise de Sequência , Homologia de Sequência de Aminoácidos , Venenos de Aranha/farmacologia , Relação Estrutura-AtividadeRESUMO
Gurmarin is a 35-residue polypeptide from the Asclepiad vine Gymnema sylvestre. It has been utilised as a pharmacological tool in the study of sweet-taste transduction because of its ability to selectively inhibit the neural response to sweet tastants in rats. We have chemically synthesised and folded gurmarin and determined its three-dimensional solution structure to high resolution using two-dimensional NMR spectroscopy. Structure calculations utilised 612 interproton-distance, 19 dihedral-angle, and 18 hydrogen-bond restraints. The structure is well defined for residues 3-34, with backbone and heavy atom rms differences of 0.27 +/- 0.09 A and 0.73 +/- 0.09 A, respectively. Gurmarin adopts a compact structure containing an antiparallel beta-hairpin (residues 22-34), several well-defined beta-turns, and a cystine-knot motif commonly observed in toxic and inhibitory polypeptides. Despite striking structural homology with delta-atracotoxin, a spider neurotoxin known to slow the inactivation of voltage-gated Na+ channels, we show that gurmarin has no effect on a variety of voltage-sensitive channels.
Assuntos
Proteínas de Plantas/química , Sequência de Aminoácidos , Dissulfetos/química , Ligação de Hidrogênio , Canais Iônicos/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/farmacologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Paladar/efeitos dos fármacosRESUMO
Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel beta-sheet comprising residues 15-18 and 37-40 and a small 3(10) helix spanning residues 10-12. The overall three-dimensional fold is similar to that of beta-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthus neurotoxin I). However, the side chains known to be functionally important in beta-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.
Assuntos
Proteínas/química , Peçonhas/química , Sequência de Aminoácidos , Animais , Defensinas , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Ornitorrinco , Conformação Proteica , Proteínas/isolamento & purificação , Proteínas/toxicidade , Ratos , Homologia de Sequência de Aminoácidos , Soluções , Ducto Deferente/efeitos dos fármacos , Peçonhas/toxicidadeAssuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Sequência de Aminoácidos , Isótopos de Carbono , Proteínas de Ciclo Celular , Divisão Celular , Escherichia coli/citologia , Escherichia coli/metabolismo , Hidrogênio , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Software , Dedos de ZincoRESUMO
Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular , Dicroísmo Circular , Dimerização , Escherichia coli/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , UltracentrifugaçãoRESUMO
DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork.
Assuntos
Bacillus subtilis/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Mutagênese Sítio-Dirigida , Replicon , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Dimerização , Genes Bacterianos/genética , Meia-Vida , Cinética , Peso Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Dobramento de Proteína , Sequências Reguladoras de Ácido Nucleico/genética , Replicon/genéticaRESUMO
Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. We have investigated the interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA). We demonstrate that this interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. We map the contact domains to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy we identify key residues within the GATA-1 N-finger which are required for FOG binding. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Primers do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Cobalto/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Espectrofotometria UltravioletaRESUMO
Mobile lipids detected using 1H-NMR in stimulated lymphocytes were correlated with cell cycle phase, expression of the interleukin-2 receptor alpha and proliferation to assess the activation status of the lymphocytes. Mobile lipid levels, IL-2R alpha expression and proliferation increased after treatment with PMA and ionomycin. PMA or ionomycin stimulation alone induced increased IL-2R alpha expression but not proliferation. PMA- but not ionomycin-stimulation generated mobile lipid. Treatment with anti-CD3 antibody did not increase IL-2R alpha expression or proliferation but did generate increased amounts of mobile lipid. The cell cycle status of thymocytes treated with anti-CD3, PMA or ionomycin alone indicated an accumulation of the cells in the G1 phase of the cell cycle. The generation of mobile lipid was abrogated in anti-CD3 antibody-stimulated thymic lymphocytes but not in splenic lymphocytes, using a phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor which blocked cells in the G1/S phase of the cell cycle. This suggests that the 1H-NMR-detectable mobile lipid may be generated in anti-CD3 antibody-stimulated thymic lymphocytes by the action of PC-PLC activity via the catabolism of PC, in the absence of classical signs of activation.