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1.
Anal Biochem ; 627: 114261, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34043980

RESUMO

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-ß, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.


Assuntos
Antineoplásicos/farmacologia , Bacillus/química , Bacteriocinas/farmacologia , Peixes/microbiologia , Neoplasias Pulmonares/metabolismo , Magaininas/farmacologia , Células A549 , Animais , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Magaininas/química , Magaininas/isolamento & purificação , Peso Molecular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Fator de Crescimento Transformador beta/metabolismo
2.
Sci Rep ; 10(1): 11652, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669585

RESUMO

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Etanolaminas/química , Magaininas/química , Fosfatidilcolinas/química , Proteínas de Xenopus/química , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Compostos de Boro/química , Membrana Celular/efeitos dos fármacos , Combinação de Medicamentos , Sinergismo Farmacológico , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Magaininas/isolamento & purificação , Magaininas/farmacologia , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Ligação Proteica , Pele/química , Espectrometria de Fluorescência , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/farmacologia , Xenopus laevis
3.
Regul Pept ; 194-195: 63-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25447193

RESUMO

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 µM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 µM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.


Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Magaininas/farmacologia , Boca/efeitos dos fármacos , Boca/microbiologia , Proteínas de Xenopus/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/isolamento & purificação , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fusobacterium nucleatum/efeitos dos fármacos , Interleucina-8/biossíntese , Magaininas/química , Magaininas/isolamento & purificação , Testes de Sensibilidade Microbiana , Boca/citologia , Porphyromonas gingivalis/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pele/química , Pele/metabolismo , Streptococcus mutans/efeitos dos fármacos , Relação Estrutura-Atividade , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/isolamento & purificação
4.
Biotechnol Prog ; 29(1): 17-22, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23125137

RESUMO

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.


Assuntos
Magaininas/genética , Magaininas/isolamento & purificação , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Xenopus laevis/genética , Xenopus laevis/metabolismo
5.
Biochimie ; 95(2): 429-35, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23142129

RESUMO

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal ß cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 µM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 µM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 µM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca(2+) concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.


Assuntos
Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/agonistas , Fragmentos de Peptídeos/farmacologia , Pipidae/metabolismo , Precursores de Proteínas/farmacologia , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Ceruletídeo/isolamento & purificação , Hipoglicemiantes/isolamento & purificação , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , L-Lactato Desidrogenase/análise , Magaininas/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Ratos , Pele/química , Pele/metabolismo , Relação Estrutura-Atividade , Proteínas de Xenopus/isolamento & purificação
6.
Artigo em Inglês | MEDLINE | ID: mdl-21498136

RESUMO

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid frog S. paratropicalis and a second polyploidization within the Xenopus lineage has produced the octoploid frog X. andrei. Peptidomic analysis of norepinephrine-stimulated skin secretions of S. paratropicalis and X. andrei led to identification of multiple peptides with growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization demonstrated that the S. paratropicalis components comprised three peptides belonging to the caerulein-precursor fragment family (CPF-SP1, -SP2 and -SP3), two peptides from the xenopsin-precursor fragment family (XPF-SP1 and -SP2), and one peptide orthologous to peptide glycine-leucine-amide (PGLa-SP1). The CPF peptides showed potent, broad-spectrum antimicrobial activity. The X. andrei components comprised two peptides from the magainin family, (magainin-AN1 and -AN2), two from the XPF family (XPF-AN1 and -AN2), two from the PGLa family(PGLa-AN1 and -AN2), and one caerulein-precursor fragment (CPF-AN1).The primary structures of these peptides indicate a close phylogenetic relationship between X. andrei and the octoploid frog X. amieti. Under the same experimental conditions, seven orthologous antimicrobial peptides were previously isolated from the diploid frog S. tropicalis, nine from the tetraploid frog X. borealis, and five from the tetraploid frog X. clivii. The data indicate, therefore, that nonfunctionalization (gene deletion) has been the most common fate of duplicated antimicrobial peptide genes following polyploidization events in the Silurana and Xenopus lineages.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Pele/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Feminino , Magaininas/química , Magaininas/isolamento & purificação , Magaininas/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Poliploidia , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/farmacologia , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/farmacologia
7.
Peptides ; 31(6): 989-94, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20226221

RESUMO

The Volcano clawed frog Xenopus amieti Kobel, du Pasquier, Fischberg, and Gloor, 1980, with a chromosome number of 2n=72, is believed to have undergone two rounds of genome duplication since evolving from a diploid ancestor. Nine peptides with differential antimicrobial activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of X. amieti that showed structural similarity to peptides previously isolated from the tetraploid frog X. laevis (2n=36) and the diploid frog Silurana (formerly Xenopus) tropicalis (2n=20). Two peptides (magainin-AM1 and -AM2) are othologous to the magainins, two peptides (PGLa-AM1 and -AM2) orthologous to peptide glycine-leucine-amide, four peptides (CPF-AM1, -AM2, -AM3, -AM4) orthologous to caerulein-precursor fragments, and one peptide (XPF-AM1) structurally similar to xenopsin-precursor fragments were characterized. CFP-AM1 (GLGSVLGKALKIGANLL.NH(2)) was the most potent peptide present in the secretions and magainin-AM2 (GVSKILHSAGKFGKAFLGEIMKS) was the most abundant. The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage. However, the very low antimicrobial activity of the magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).


Assuntos
Magaininas/isolamento & purificação , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Anti-Infecciosos/análise , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Escherichia coli/efeitos dos fármacos , Magaininas/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Norepinefrina , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Pele/química , Pele/efeitos dos fármacos , Pele/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Xenopus
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