RESUMO
The adaptation of amphibians to diverse environments is closely related to the characteristics of their skin. The complex glandular system of frog skin plays a pivotal role in enabling these animals to thrive in both aquatic and terrestrial habitats and consists of crucial functions such as respiration and water balance as well as serving as a defensive barrier due to the secretion of bioactive compounds. We herein report the first investigation on the skin secretion of Odontophrynus americanus, as a potential source of bioactive peptides and also as an indicator of its evolutionary adaptations to changing environments. Americanin-1 was isolated and identified as a neutral peptide exhibiting moderate antibacterial activity against E. coli. Its amphipathic sequence including 19 amino acids and showing a propensity for α-helix structure is discussed. Comparisons of the histomorphology of the skin of O. americanus with other previously documented species within the same genus revealed distinctive features in the Patagonian specimen, differing from conspecifics from other Argentine provinces. The presence of the Eberth-Katschenko layer, a prevalence of iridophores, and the existence of glycoconjugates in its serous glands suggest that the integument is adapted to retain skin moisture. This adaptation is consistent with the prevailing aridity of its native habitat.
Assuntos
Anuros , Pele , Animais , Pele/química , Escherichia coli/efeitos dos fármacos , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Estrutura Molecular , Argentina , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
Peptides modulate many processes of human physiology targeting ion channels, protein receptors, or enzymes. They represent valuable starting points for the development of new biologics against communicable and non-communicable disorders. However, turning native peptide ligands into druggable materials requires high selectivity and efficacy, predictable metabolism, and good safety profiles. Machine learning models have gradually emerged as cost-effective and time-saving solutions to predict and generate new proteins with optimal properties. In this chapter, we will discuss the evolution and applications of predictive modeling and generative modeling to discover and design safe and effective antimicrobial peptides. We will also present their current limitations and suggest future research directions, applicable to peptide drug design campaigns.
Assuntos
Peptídeos Antimicrobianos , Produtos Biológicos , Humanos , Inteligência Artificial , Aprendizado de Máquina , Desenho de FármacosRESUMO
Amphibians' skin is a rich source of natural antimicrobial peptides (AMPs). These AMPs exhibit marked inter- and intraspecific sequence divergence linked to the arms race between host and pathogens. Here, we combine peptidomics, molecular modeling, and phylogenetic analyses to understand the evolution of AMPs in Cophomantini, a diverse clade of neotropical tree frogs, and to investigate their interaction with bacterial membranes. Consistent with results in other amphibians, all species of Cophomantini secrete a mixture of peptides. We selected the hylin peptide family to survey sequence variability and the presence of common amino acid motifs. We found that most species secrete a unique set of hylins that, though variable, share the conserved motif Gly-X-X-X-Pro-Ala-X-X-Gly, with Gly and Pro colocalizing with charged or polar residues. Our modeling revealed that Pro curves the peptide through a hinge, facilitating its insertion into the bacterial membrane and, once inserted, contributes to stabilizing the pore structure. The phylogenetic inference using hylid prepro-peptides showed the need to classify new AMPs using the full-length sequence of the prepro-peptide region and highlighted the complex relationships between peptide families. Our findings revealed that conserved motifs occurred independently in distinct AMP families, suggesting a convergent evolution and a significant role in peptide-membrane interactions.
Assuntos
Peptídeos Antimicrobianos , Peptídeos , Humanos , Animais , Sequência de Aminoácidos , Filogenia , Peptídeos/química , Anuros/metabolismoRESUMO
Amphibians have a great diversity of bioactive peptides in their skin. The cDNA prepro-peptide sequencing allowed the identification of five novel mature peptides expressed in the skin of Boana pulchella, four with similar sequences to hylin peptides having a cationic amphipathic-helical structure. Whole mature peptides and some of their fragments were chemically-synthesized and tested against Gram-positive and Gram-negative bacterial strains. The mature peptide hylin-Pul3 was the most active, with a MIC= 14 µM against Staphylococcus aureus. Circular dichroism assays indicated that peptides are mostly unstructured in buffer solutions. Still, adding large unilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoylphosphatidylglycerol increased the α-helix content of novel hylins. These results demonstrate the strong influence of the environment on peptide conformation and highlight its significance while addressing the pharmacology of peptides and their biological function in frogs.
Assuntos
Anuros , Peptídeos , Animais , Sequência de Aminoácidos , Peptídeos/farmacologia , Peptídeos/química , Lipídeos , Dicroísmo CircularRESUMO
Bombesin mediates several biological activities in the gastrointestinal (GI) tract and central nervous system in mammals, including smooth muscle contraction, secretion of GI hormones and regulation of homeostatic mechanisms. Here, we report a novel bombesin-like peptide isolated from Boana raniceps. Its amino acid sequence, GGNQWAIGHFM-NH2, was identified and structurally confirmed by HPLC, MS/MS and 454-pyrosequencing; the peptide was named BR-bombesin. The effect of BR-bombesin on smooth muscle contraction was assessed in ileum and esophagus, and its anti-secretory activity was investigated in the stomach. BR-bombesin exerted significant contractile activity with a concentration-response curve similar to that of commercially available bombesin in ileum strips of Wistar rats. In esophageal strips, BR-bombesin acted as an agonist, as many other bombesin-related peptides act, although with different behavior compared to the muscarinic agonist carbachol. Moreover, BR-bombesin inhibited stomach secretion by approximately 50% compared to the untreated control group. This novel peptide has 80% and 70% similarity with the 10-residue C-terminal domain of human neuromedin B (NMB) and human gastrin releasing peptide (GRP10), respectively. Molecular docking analysis revealed that the GRP receptor had a binding energy equal to - 7.3 kcal.mol-1 and - 8.5 kcal.mol-1 when interacting with bombesin and BR-bombesin, respectively. Taken together, our data open an avenue to investigate BR-bombesin in disorders that involve gastrointestinal tract motility and acid gastric secretion.
Assuntos
Bombesina , Receptores da Bombesina , Animais , Anuros/metabolismo , Bombesina/metabolismo , Bombesina/farmacologia , Mamíferos/metabolismo , Simulação de Acoplamento Molecular , Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo , Estômago , Espectrometria de Massas em TandemRESUMO
The molecular bases for the symbiosis of the amphibian skin microbiome with its host are poorly understood. Here, we used the odor-producer Pseudomonas sp. MPFS and the treefrog Boana prasina as a model to explore bacterial genome determinants and the resulting mechanisms facilitating symbiosis. Pseudomonas sp. MPFS and its closest relatives, within a new clade of the P. fluoresens Group, have large genomes and were isolated from fishes and plants, suggesting environmental plasticity. We annotated 16 biosynthetic gene clusters from the complete genome sequence of this strain, including those encoding the synthesis of compounds with known antifungal activity and of odorous methoxypyrazines that likely mediate sexual interactions in Boana prasina. Comparative genomics of Pseudomonas also revealed that Pseudomonas sp. MPFS and its closest relatives have acquired specific resistance mechanisms against host antimicrobial peptides (AMPs), specifically two extra copies of a multidrug efflux pump and the same two-component regulatory systems known to trigger adaptive resistance to AMPs in P. aeruginosa. Subsequent molecular modeling indicated that these regulatory systems interact with an AMP identified in Boana prasina through the highly acidic surfaces of the proteins comprising their sensory domains. In agreement with a symbiotic relationship and a highly selective antibacterial function, this AMP did not inhibit the growth of Pseudomonas sp. MPFS but inhibited the growth of another Pseudomonas species and Escherichia coli in laboratory tests. This study provides deeper insights into the molecular interaction of the bacteria-amphibian symbiosis and highlights the role of specific adaptive resistance toward AMPs of the hosts.
Assuntos
Bactérias , Simbiose , Animais , Anuros , Bactérias/genética , Genoma Bacteriano , GenômicaRESUMO
Ocellatins are a family of antimicrobial peptides found exclusively in the Leptodactylus genus. To date, 10 species have been studied and more than 23 peptides described. Here we report the sequences of five new peptides from the skin of the frog Leptodactylus latrans (Anura: Leptodactylidae) determined by cDNA cloning of the complete prepro-peptide structures. The mature peptides were characterized with in silico tools and compared with those previously described. With 21 amino acid residues, this new set of peptides not previously described in the Leptodactylus genus share between 100 and 76.2% similarity to ocellatin antimicrobial peptides. These novel peptides are cationic and their three-dimensional (3D) structure holds the highly conserved residues G1, D4, K7, and K11 and a high theoretical amphipathic α-helix content. Furthermore, in silico analyses of these new peptides predicted antimicrobial activity. This study is framed in the context of previous work published about ocellatins, and therefore, provides a review of this intriguing family of peptides.
RESUMO
The skin glands of amphibian species hold a major component of their innate immunity, namely a unique set of antimicrobial peptides (AMPs). Although most of them have common characteristics, differences in AMP sequences allow a huge repertoire of biological activity with varying degrees of efficacy. We present the first study of the AMPs from Pleurodema somuncurence (Anura: Leptodactylidae: Leiuperinae). Among the 11 identified mature peptides, three presented antimicrobial activity. Somuncurin-1 (FIIWPLRYRK), somuncurin-2 (FILKRSYPQYY), and thaulin-3 (NLVGSLLGGILKK) inhibited Escherichia coli growth. Somuncurin-1 also showed antimicrobial activity against Staphylococcus aureus. Biophysical membrane model studies revealed that this peptide had a greater permeation effect in prokaryotic-like membranes and capacity to restructure liposomes, suggesting fusogenic activity, which could lead to cell aggregation and disruption of cell morphology. This study contributes to the characterization of peptides with new sequences to enrich the databases for the design of therapeutic agents. Furthermore, it highlights the importance of investing in nature conservation and the power of genetic description as a strategy to identify new compounds.
Assuntos
Espécies em Perigo de Extinção , Peptídeos/química , Peptídeos/farmacologia , Ranidae/metabolismo , Pele/química , Sequência de Aminoácidos , Animais , Antioxidantes/farmacologia , Argentina , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipossomos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Permeabilidade , Staphylococcus aureus/efeitos dos fármacosRESUMO
Peptides from skin secretions of amphibians are considered important components of their immune system and also play a relevant role in their defense mechanism against predators. Herein, by using mass spectrometry (MS), we characterize the sequence of 13 peptides from the gland secretion of the hylid tree frog, Boana punctata. Using in situ matrix-assisted laser desorption ionization imaging MS of a transverse section of the skin tissue, we show that some peptides are stored as longer molecules that are cleaved after being secreted, whereas others do not undergo any modification. Sequence comparison with peptides from other Boana species and analysis of the three-dimensional theoretical structure indicate that this cleavage depends on both the presence of a specific sequence motif and the secondary structure. The fact that peptides undergo a rapid cleavage upon secretion suggests that stored and secreted peptides may have distinct roles for anuran survival, including defense against pathogens and predators.
RESUMO
Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999-1005, 2018.
Assuntos
Cromatografia de Afinidade/métodos , Hormônio do Crescimento Humano/isolamento & purificação , Leite/química , Proteínas Recombinantes/isolamento & purificação , Animais , Hormônio do Crescimento Humano/química , Humanos , Análise Serial de Proteínas , Proteínas Recombinantes/químicaRESUMO
The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H]+ = 1543.69Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide.
Assuntos
Proteínas de Anfíbios/genética , Peptídeos Catiônicos Antimicrobianos/genética , Anuros/fisiologia , Infecções Bacterianas/imunologia , Fibroblastos/fisiologia , Microglia/metabolismo , Pele/metabolismo , Proteínas de Anfíbios/imunologia , Proteínas de Anfíbios/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antioxidantes/metabolismo , Clonagem Molecular , Sequestradores de Radicais Livres/metabolismo , Camundongos , Estrutura Molecular , Células NIH 3T3 , Neuroproteção , Oxirredução , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/imunologia , Anticorpos/imunologia , Glycine max/imunologia , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/imunologia , Epitopos/imunologiaRESUMO
Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 µM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 µM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively.
Assuntos
Cromatografia de Afinidade/métodos , Técnicas de Química Combinatória , Eritropoetina/isolamento & purificação , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Eritropoetina/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TermodinâmicaRESUMO
Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix.
Assuntos
Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ácido Acético/química , Acetonitrilas/química , Hidróxido de Amônia , Hidróxidos/química , Biblioteca de Peptídeos , Peptídeos/química , Análise de Sequência de Proteína , Água/químicaRESUMO
To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.
Assuntos
Técnicas de Química Combinatória/métodos , Biblioteca de Peptídeos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes , MicroesferasRESUMO
Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1-->3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution.