RESUMO
Drosomycin is a 44-residue antifungal peptide with four intramolecular disulfide bridges which have been isolated from immune-challenged Drosophila. To produce adequate amounts of this peptide for 3D-structure analysis, studies on the mode of action and activity spectrum, we expressed a synthetic cDNA in Saccharomyces cerevisiae. For this purpose, we used the mating factor alpha gene and concomitantly overexpressed the KEX2 gene to increase the yield of fully processed drosomycin. Using a combination of Edman degradation and mass spectrometry, we show that drosomycin shares the same array of intramolecular disulfide bridges than plant defensins, in addition to their sequence similarities.
Assuntos
Antifúngicos/química , Dissulfetos/química , Proteínas de Drosophila , Drosophila melanogaster/química , Proteínas de Insetos , Pró-Proteína Convertases , Proteínas/química , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Antifúngicos/isolamento & purificação , Sequência de Bases , DNA Recombinante , Expressão Gênica , Vetores Genéticos/genética , Fator de Acasalamento , Dados de Sequência Molecular , Peso Molecular , Peptídeos/genética , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/genética , Subtilisinas/genéticaRESUMO
Antimicrobial peptides constitute an important component of the mammalian innate immune response. Several types of antimicrobial peptides, including the beta-defensins, are produced at epithelial surfaces in response to infectious threats. Here we show that a class of small molecules, including l-isoleucine and several of its analogs, can specifically induce epithelial beta-defensin expression. This induction is transcriptional in nature and involves activation of the NF-kappaB/rel family of trans-activating factors. We hypothesize that these substances represent unique markers for the presence of pathogens and are recognized by innate immune pattern recognition receptors. Isoleucine or its analogs ultimately may have clinical utility as novel immunostimulants that could bolster the barrier defenses of mucosal surfaces.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Isoleucina/farmacologia , Transcrição Gênica/efeitos dos fármacos , beta-Defensinas/genética , Animais , Bovinos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Isoleucina/análogos & derivados , Isoleucina/química , Camundongos , Estrutura Molecular , NF-kappa B/metabolismoRESUMO
In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense.
Assuntos
Antifúngicos , Proteínas de Drosophila , Drosophila/imunologia , Proteínas de Insetos , Biossíntese Peptídica , Plantas/imunologia , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/imunologia , Precursores de Proteínas/genética , Proteínas/imunologia , Proteínas/farmacologia , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We have isolated, from the hemolymph of unchallenged scorpions of the species Androctonus australis, three distinct antimicrobial peptides, which we have fully characterized by Edman degradation, electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. Two are novel molecules: (i) androctonin, a 25-residue peptide with two disulfide bridges, active against both bacteria (Gram-positive and Gram-negative) and fungi and showing marked sequence homology to tachyplesins and polyphemusins from horseshoe crabs; and (ii) buthinin, a 34-residue antibacterial (Gram-positive and Gram-negative) peptide with three disulfide bridges. The third peptide contains 37 residues and three disulfide bridges and clearly belongs to the family of anti-Gram-positive insect defensins. We have synthesized androctonin and explored its activity spectrum and mode of action.
Assuntos
Antibacterianos/química , Cisteína/análise , Hemolinfa/química , Peptídeos , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Hemólise/efeitos dos fármacos , Espectrometria de Massas , Microscopia Eletrônica , Dados de Sequência Molecular , EscorpiõesRESUMO
Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.