RESUMO
The phosphatidylethanolamine binding proteins (PEBPs) family is evolutionarily conserved and involved in different physiological phenomena. PEBPs were found in many species from bacteria to mammals. Despite numerous studies, PEBPs' biological function and mode of action remain elusive. Based on sequence homology, seven PEBP genes were detected in the Drosophila genome. Only one of them, the odorant binding protein (OBP), has been characterized. To date nothing is known concerning the expression pattern and biological roles of the six other PEBP genes. By RT-PCR and Western blot analysis, we examined expression of the PEBPs in different tissues and embryos. The 6 PEBPs were differentially expressed. Only one, CG10298, is specific of only one tissue: the testis. Additionally, by comparing in wild type and male-sterile mutants we show that CG10298 is present only during spermatid differentiation. Furthermore, by comparing structural parameters of the six PEBP proteins with those of human PEBP-1, we have established that PEBP CG10298 is most closely related to human PEBP.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Testículo/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologia , Masculino , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/classificação , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , RNA/análise , Homologia de Sequência , Espermátides/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Distribuição TecidualRESUMO
In an attempt to increase the antimicrobial activity of the insect defensin from Anopheles gambiae, which is active against Staphylococcus aureus at low concentration, hybrid defensins were designed by combining conserved sequence regions and variable regions of insect defensins. Their activity against S. aureus strains sensitive and resistant to conventional antibiotics was evaluated, and the toxicity of the most active molecules was tested. The three-dimensional structure of Anopheles gambiae defensin and five hybrids were determined by NMR and molecular modelling. This strategy led to the design of two chimeric defensins with increased activity compared with the native molecule, but one of them appears to be toxic to mice at a rather low concentration. The structure of the CS alphabeta motif, which is a characteristic of insect defensin, is sensitive to sequence modifications, in particular in the N-terminal loop. The existence of the CS alphabeta is most probably a prerequisite for the stability and the activity of the molecule, but is not sufficient by itself since the hybrid displaying the best defined structure is not active against the tested strains. The analysis of the structure, in relation with the activity and the toxicity data, underlines the importance of turns and of the N-terminal loop. Residues located in the turns contributing to the preservation of positive electrostatic areas at the surface of the molecules seem particularly important for the activity of the molecule, while residues involved in the N-terminal loop are both involved in the modulation of the activity and the toxicity of the molecule.
Assuntos
Anti-Infecciosos/farmacologia , Defensinas/farmacologia , Desenho de Fármacos , Peptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Defensinas/química , Interações Hidrofóbicas e Hidrofílicas , Insetos/química , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Eletricidade Estática , Propriedades de Superfície/efeitos dos fármacos , Testes de ToxicidadeRESUMO
Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.
Assuntos
Proteínas de Transporte/química , Nicotiana , Proteínas de Plantas/química , Sequência de Aminoácidos , Antígenos de Plantas , Simulação por Computador , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Isótopos de Nitrogênio , Conformação ProteicaRESUMO
Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Nicotiana/metabolismo , Antígenos de Plantas , Sequência de Bases , Fenômenos Biomecânicos , Proteínas de Transporte/genética , Primers do DNA , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Isótopos de Nitrogênio , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluções , Nicotiana/químicaAssuntos
Drosophila melanogaster/química , Proteína de Ligação a Fosfatidiletanolamina/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Soluções , Homologia Estrutural de ProteínaRESUMO
The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an alpha-helical segment (Phe4-Gln14) and a two-stranded (Phe19-Asp25 and Gln28-Phe33) antiparallel beta-sheet forming a "cysteine stabilized alphabeta motif" (CSalphabeta) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD-1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS-PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]
Assuntos
Antibacterianos/química , Anti-Infecciosos/química , Antifúngicos/química , Proteínas de Drosophila , Hemócitos/química , Isópteros/química , Peptídeos , Animais , Peptídeos Catiônicos Antimicrobianos , Cisteína/química , Defensinas/química , Proteínas de Insetos/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos Policíclicos/química , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , SoluçõesRESUMO
Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.
Assuntos
Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Desenho de Fármacos , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Clonagem Molecular , Cryptococcus neoformans/efeitos dos fármacos , Fusarium/efeitos dos fármacos , Hemolinfa/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Larva/química , Lepidópteros/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Scedosporium/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletricidade Estática , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
UDP-GalNAc:polypeptide alphaN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr. Structural features underlying their enzymatic activity and their specificity are still unidentified. In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1. Fold recognition methods identified as appropriate templates the bovine alpha1,3galactosyltransferase and the human alpha1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ cation were docked. UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193. The ribose is found in van der Waals contacts with F124 and L189. The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126. The Mn2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme. Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast. Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.
Assuntos
Substituição de Aminoácidos/genética , Modelos Químicos , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Mutação Puntual/genética , Uridina Difosfato N-Acetilgalactosamina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Bovinos , Expressão Gênica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , N-Acetilgalactosaminiltransferases/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Serina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Treonina/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismoRESUMO
Tfs1p and Ylr179cp are yeast proteins belonging to the PEBP family. Tfs1p, but not Ylr179cp, has been shown to interact with and inhibit Ira2p, a GTPase-activating protein of Ras. Tfs1p has been shown to be a specific inhibitor of the CPY protease and the 3D structure of the complex has been resolved. To shed light on the molecular determinants of Tfs1p involved in the Tfs1/Ira2 interaction, the 3D structure of Ylr179cp has been modelled and compared to that of Tfs1p. Tfs1p point mutants and Tfs1 hybrid proteins combining regions of Tfs1p and Ylr179cp were also designed and their function was tested. Results, interpreted from a structural point of view, show that the accessibility of the surface pocket of Tfs1p, its N-terminal region and the specific electrostatic properties of a large surface region containing these two elements, play a crucial role in this interaction.
Assuntos
Proteínas Ativadoras de GTPase/química , Modelos Moleculares , Engenharia de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Bases , Primers do DNA , Proteínas Ativadoras de GTPase/metabolismo , Imunoprecipitação , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a beta-hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 microM they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3)), Thr(6,11,15), D-Pro(9)]-Gm with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr(2,6,11,15), Pro(8)]-D-Gm and [Thr(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was further analyzed by NMR spectroscopy. (1)H NMR experiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-D-Gm presents a conformation very similar to that of Gm. In our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Candida albicans/efeitos dos fármacos , Dicroísmo Circular , Dissulfetos/química , Desenho de Fármacos , Estabilidade de Medicamentos , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Prolina/química , Conformação Proteica , Staphylococcus aureus/efeitos dos fármacos , Eletricidade EstáticaRESUMO
Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Muscidae/química , Peptídeos/química , Trifluoretanol/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Soluções/químicaRESUMO
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFkappaB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly beta-sheets at pH 7. The good dispersion of the crosspeaks on the 1H-15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/isolamento & purificação , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin consists in a well-resolved two-stranded antiparallel betasheet connected by a noncanonical betaturn. A comparison between the structures of gomesin and protegrin-1 from porcine and androctonin from scorpion outlines several common features in the distribution of hydrophobic and hydrophilic residues. The N- and C-termini, the betaturn and one face of the betasheet are hydrophilic, but the hydrophobicity of the other face depends on the peptide. The similarities suggest that the molecules interact with membranes in an analogous manner. The importance of the intramolecular disulfide bridges in the biological activity of gomesin is being investigated.
Assuntos
Anti-Infecciosos/química , Proteínas Sanguíneas/química , Aranhas/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Cistina , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
Recently two beta-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053-51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel beta-sheet stabilized by three disulfide bridges with a pairing typical of beta-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian beta-defensins showed that the hydrophobic patch is not preserved in mammalian beta-defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids.
Assuntos
Mucosa Gástrica/metabolismo , beta-Defensinas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aves , Cátions , Cisteína/química , Magnésio/química , Cloreto de Magnésio/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sais/farmacologia , Homologia de Sequência de Aminoácidos , Sódio/químicaRESUMO
PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels.
Assuntos
Albuminas/química , Endotoxinas/química , Modelos Moleculares , Pisum sativum/química , Proteínas de Plantas/química , Albuminas/isolamento & purificação , Albuminas/toxicidade , Sequência de Aminoácidos , Animais , Besouros/efeitos dos fármacos , Simulação por Computador , Dissulfetos/química , Endotoxinas/isolamento & purificação , Endotoxinas/toxicidade , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/toxicidade , Dobramento de Proteína , Prótons , Sementes/químicaRESUMO
The gut epithelium is an essential interface in insects that transmit parasites. We investigated the role that local innate immunity might have on vector competence, taking Stomoxys calcitrans as a model. S. calcitrans is sympatric with tsetse flies, feeds on many of the same vertebrate hosts, and is thus regularly exposed to the trypanosomes that cause African sleeping sickness and nagana. Despite this, S. calcitrans is not a cyclical vector of these trypanosomes. Trypanosomes develop exclusively in the lumen of digestive organs, and so epithelial immune mechanisms, and in particular antimicrobial peptides (AMPs), may be the prime determinants of the fate of an infection. To investigate why S. calcitrans is not a cyclical vector of trypanosomes, we have looked in its midgut for AMPs with trypanolytic activity. We have identified a new AMP of 42 amino acids, which we named stomoxyn, constitutively expressed and secreted exclusively in the anterior midgut of S. calcitrans. It displays an amphipathic helical structure and exhibits a broad activity spectrum affecting the growth of microorganisms. Interestingly, this AMP exhibits trypanolytic activity to Trypanosoma brucei rhodesiense. We argue that stomoxyn may help to explain why S. calcitrans is not a vector of trypanosomes causing African sleeping sickness and nagana.
Assuntos
Anti-Infecciosos/farmacologia , Epitélio/imunologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , DNA Complementar/metabolismo , Dípteros , Relação Dose-Resposta a Droga , Feminino , Fungos/efeitos dos fármacos , Biblioteca Gênica , Mucosa Intestinal/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Fatores de Tempo , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
The hydrophobic cavity of Lipid Transfer Protein 1 from Nicotiana tabacum is investigated in detail by NMR using xenon as a spy. The analysis of the (129)Xe chemical shifts and self-relaxation times gives evidence of protein-xenon interaction. Thermodynamics of the binding is characterized through the study of aliphatic (1)H and (13)C chemical shift variation as a function of xenon pressure. The binding constant is evaluated to 75.5 +/- 1.0 M(-1) at 293 K. The location of xenon inside the cavity is deduced from SPINOE experiments. The noble gas appears to occupy four sites, and xenon self-relaxation experiments indicate that it quickly jumps between different sites. The chemical shifts of amide protons and nitrogens also depend on the xenon concentration, either specifically or nonspecifically for atoms at the external surface of the protein. Yet, contrary to aliphatic atoms, they do not correspond to short-range interactions as confirmed by magnetization transfer experiments between laser-polarized xenon and protons in H(2)O. These (15)N chemical shift variations, used in combination with (15)N transverse self-relaxation rates to determine the lower limit of the binding rate, consequently reveal subtle changes in the structure of the protein upon binding.
Assuntos
Proteínas de Transporte/química , Nicotiana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Plantas/química , Antígenos de Plantas , Proteínas de Transporte/metabolismo , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Proteínas de Plantas/metabolismo , Soluções , Termodinâmica , XenônioRESUMO
Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from the coleopteran Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequence identity above 80% and active against the Candida glabrata yeast strain were identified. Alo-3 displayed the highest activity against Candida glabrata and was thus chosen for structure determination using NMR spectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges. The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software, allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structural features characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is the first peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level of activity significantly higher against C. glabrata than Alo-1 or Alo-2. It has no negatively charged residues and displays on its surface a cationic pole that may account for its antifungal activity. This finding is validated by the comparison of the structure of Alo-3 with the structure of other structurally related peptides from other sources also showing antifungal activity.