RESUMO
There has been a growing interest in developing natural antioxidants with high efficiency and low cost. Bioactive protein hydrolysates could be a potential source of natural and safer antioxidants. The objectives of this study were to hydrolyze corn gluten meal using three plant-derived proteases, namely papain, ficin, and bromelain, to produce antioxidative hydrolysates and peptides and to characterize the antioxidant performances using both chemical assays and a ground meat model. The optimum hydrolysis time for papain was 3 h, and for ficin and bromelain was 4 h. The hydrolysates were further separated by sequential ultrafiltration to 5 hydrolysate fractions named F1 to F5 from low molecular weight (MW) (<1 kDa) to high MW range (>10 kDa), which were further characterized for TPC, free radical scavenging capacity against DPPH and ABTS, and metal chelating activity. The fraction F4 produced by papain (CH-P4), F1 produced by ficin (CH-F1), and F3 produced by bromelain (CH-B3) showed the strongest antioxidant activity and yield, respectively. These three fractions were incorporated into ground pork to determine their inhibition effects on lipid oxidation during a 16-day storage period. The inhibition effect was enhanced with the addition of higher amount of hydrolysate (e.g., 1000 vs. 500 mg/kg). The CH-P4 reduced lipid oxidation in ground meat by as much as 30.45%, and CH-B3 reduced oxidation by 27.2% at the same level, but the inhibition was only 13.83% with 1000 mg/kg of CH-F1. The study demonstrated that CGM protein hydrolysates and peptides could be used as naturally derived antioxidant in retarding lipid oxidation and improving product storage stability.
Assuntos
Antioxidantes/química , Glutens/química , Biossíntese Peptídica , Hidrolisados de Proteína/biossíntese , Zea mays/química , Bromelaínas , Ficina , Hidrólise , Cinética , Peroxidação de Lipídeos , Oxirredução , Papaína/química , FenolRESUMO
Angiotensin I-converting enzyme (ACE) is a paramount therapeutic target to treat hypertension. ACE inhibitory peptides derived from food protein sources are regarded as safer alternatives to synthetic antihypertensive drugs for treating hypertension. Recently, marine organisms have started being pursued as sources of potential ACE inhibitory peptides. Marine organisms such as fish, shellfish, seaweed, microalgae, molluscs, crustaceans, and cephalopods are rich sources of bioactive compounds because of their high-value metabolites with specific activities and promising health benefits. This review aims to summarize the studies on peptides from different marine organisms and focus on the potential ability of these peptides to inhibit ACE activity.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Organismos Aquáticos/metabolismo , Biossíntese Peptídica , Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Biomarcadores , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Peptídeos/químicaRESUMO
The prebiotic replication of information-coding molecules is a central problem concerning life's origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH2 yields the isotactic product FRFRFRFR-NH2, 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH2 and Ac-VDVDVDVDV-NH2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.
Assuntos
Aminoácidos/química , Amiloide/química , Técnicas de Química Sintética/métodos , Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Amiloide/metabolismo , Amiloide/ultraestrutura , Arginina/química , Arginina/genética , Arginina/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Origem da Vida , Biossíntese Peptídica/genética , Peptídeos/genética , Peptídeos/metabolismo , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Cloreto de Sódio/química , Estereoisomerismo , Temperatura , Moldes GenéticosRESUMO
Herein, we present the design and synthesis of a catalytically active peptide-nanoparticle conjugate whose activity is regulated by a defined conformational change in the self-assembled peptide monolayer. A catalytically active peptide, designed after the heterodimeric α-helical coiled-coil principle was immobilized onto gold nanoparticles, and kinetic studies were performed according to the Michaelis-Menten model. The formed peptide monolayer at the gold nanoparticle surface accelerated p-nitrophenylacetate (pNPA) hydrolysis by 1 order of magnitude compared to the soluble peptide while exhibiting no defined secondary structure as determined by infrared (IR) and circular dichroism (CD) spectroscopy. Addition of the complementary peptide-induced coiled-coil formation while significantly hindering the pNPA hydrolysis catalyzed by the peptide-nanoparticle conjugate. The heptad repeat sequence of a coiled-coil opens up the opportunity for regulation of conformation and thus catalytic activity of peptide-nanoparticle conjugates upon interaction with a complementary coiled-coil sequence. Strategies of regulation of catalytic activity by interaction with a complementary cofactor/ligand are well-established in nature and are introduced here into rationally designed peptide-nanoparticle conjugates.
Assuntos
Aminoácidos/química , Nanopartículas Metálicas/química , Biossíntese Peptídica , Peptídeos/síntese química , Catálise , Ouro/química , Hidrólise , Peptídeos/química , Fenilacetatos/química , Estrutura Secundária de ProteínaRESUMO
Los péptidos bioactivos o péptidos con actividad biológica producidos durante la digestión gastrointestinal o la elaboración de alimentos pueden ejercer un importante papel en la regulación y la modulación metabólica, que sugiere su uso potencial como nutracéuticos e ingredientes de alimentos funcionales para promoción de la salud y la reducción del riesgo de enfermedad. En los últimos años se han destinado muchos esfuerzos al estudio de las diferentes actividades beneficiosas que estos péptidos bioactivos pueden tener sobre el organismo, incluyendo su actividad antihipertensiva, hipocolesterolemiante, antioxidante, antimicrobianae inmunomoduladora, así como su efecto opiáceo. Así mismo se están destinando esfuerzos en investigación para la detección de fuentes alimentarias de péptidos bioactivos así como al estudio de su biodisponibilidad, de sus propiedades funcionales y de sus mecanismos de acción (AU)
Bioactive peptides, or peptides with biological activity produced during gastrointestinal digestion or food processing, could play an important role in metabolic regulation and modulation, suggesting their potential use as nutraceuticals and ingredients of functional foods to promote health and reduce the risk of disease. In the last few years, efforts have been made to study the various potential beneficial activities of these bioactive peptides in the body, including their antihypertensive, hypocholesterolemic, antioxidant, antimicrobial, immunomodulatory, and opiate-like activities. Likewise, research is currently being carried out to detect food sources of bioactive peptides as well as to study their bioavailability, functional properties and mechanisms of action (AU)
Assuntos
Humanos , Peptídeos/fisiologia , Digestão/fisiologia , Suplementos Nutricionais , Anticolesterolemiantes/metabolismo , Biossíntese Peptídica/fisiologia , Dietoterapia/métodosRESUMO
Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.
Assuntos
Proteínas Fúngicas/metabolismo , Metaloproteases/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Pseudallescheria/metabolismo , Caseínas/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Hemoglobinas/metabolismo , Humanos , Imunoglobulina G/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/biossíntese , Metaloproteases/química , Peso Molecular , Micetoma/metabolismo , Micetoma/microbiologia , Biossíntese Peptídica , Peptídeos/antagonistas & inibidores , Peptídeos/química , Fenantrolinas/farmacologia , Scedosporium/metabolismo , Albumina Sérica/metabolismoRESUMO
Methods for the incorporation of non-natural amino acids into proteins have advanced significantly over recent years and in this tutorial review we aim to give a general overview of the area. These techniques offer the possibility of modulating the structures and functions of proteins and thus permit the generation of novel designed systems for both biocatalytic and mechanistic studies. Four complementary approaches are discussed in detail along with examples of their application. The advantages and disadvantages of each technique are also discussed.
Assuntos
Aminoácidos/química , Peptídeos/química , Peptídeos/síntese química , Engenharia de Proteínas , Proteínas/química , Proteínas/síntese química , Biossíntese Peptídica , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismoRESUMO
The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.
Assuntos
Glicina/química , Leucina/química , Peptídeos/química , Eletricidade Estática , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Anuros , Sequência de Bases , Encéfalo/metabolismo , Cátions , Membrana Celular/metabolismo , Dicroísmo Circular , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/metabolismo , Eritrócitos/metabolismo , Escherichia coli/metabolismo , Hemólise , Mucosa Intestinal/metabolismo , Cinética , Bicamadas Lipídicas/química , Modelos Químicos , Dados de Sequência Molecular , Biossíntese Peptídica , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Filogenia , Conformação Proteica , Estrutura Secundária de Proteína , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Pele/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Distribuição TecidualRESUMO
Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Canais de Cálcio/metabolismo , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/química , Cálcio/metabolismo , Cátions , Cromatografia Líquida de Alta Pressão , DNA Complementar/metabolismo , Dissulfetos , Relação Dose-Resposta a Droga , Eletrofisiologia , Hidrólise , Íons , Cinética , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Caramujos , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo , Triptofano/químicaRESUMO
A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit approximately 80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.
Assuntos
Proteínas do Tecido Nervoso/química , Neuropeptídeos/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Bases de Dados como Assunto , Relação Dose-Resposta a Droga , Etiquetas de Sequências Expressas , Genoma Humano , Humanos , Hipotálamo/metabolismo , Hibridização In Situ , Masculino , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Biossíntese Peptídica , RNA Mensageiro/metabolismo , Ranidae , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
OBJECTIVE: This study was conducted to examination whether Ginkgo biloba extract (GBE), a Chinese herb with antioxidant activity, could reduce cytokine-induced monocyte/human aortic endothelial cell (HAEC) interaction, a pivotal early event in atherogenesis. METHODS AND RESULTS: Pretreatment of HAECs with GBE (50 and 100 microg/mL for 18 hours) significantly suppressed cellular binding between the human monocytic cell line U937 and tumor necrosis factor-alpha (TNF-alpha)-stimulated HAECs by using in vitro binding assay (68.7% and 60.1% inhibitions, respectively). Cell enzyme-linked immunosorbent assay and immunoblot analysis showed that GBE (50 microg/mL for 18 hours) significantly attenuated TNF-alpha-induced cell surface and total protein expression of vascular cellular adhesion molecule-1 and intracellular adhesion molecule-1 (63.5% and 69.2%, respectively; P<0.05). However, pretreatment with probucol (5 micromol/L for 18 hours) reduced the expression of vascular cellular adhesion molecule-1 but not intracellular adhesion molecule-1. Preincubation of HAECs with GBE or probucol significantly reduced intracellular reactive oxygen species formation induced by TNF-alpha (76.8% and 68.2% inhibitions, respectively; P<0.05). Electrophoretic mobility shift assay demonstrated that both GBE and probucol inhibited transcription factor nuclear factor-kappaB activation in TNF-alpha-stimulated HAECs (55.2% and 65.6% inhibitions, respectively) but only GBE could inhibit the TNF-alpha-stimulated activator protein 1 activation (45.1% inhibition, P<0.05). CONCLUSIONS: GBE could reduce cytokine-stimulated endothelial adhesiveness by downregulating intracellular reactive oxygen species formation, nuclear factor-kappaB and activator protein 1 activation, and adhesion molecule expression in HAECs, supporting the notion that the natural compound Ginkgo biloba may have potential implications in clinical atherosclerosis disease.
Assuntos
Moléculas de Adesão Celular/biossíntese , Células Epiteliais/efeitos dos fármacos , Ginkgo biloba/química , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Aorta/citologia , Arteriosclerose/prevenção & controle , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , NF-kappa B/fisiologia , Biossíntese Peptídica/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/uso terapêutico , Fator de Transcrição AP-1/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Células U937/efeitos dos fármacos , Células U937/metabolismoRESUMO
Peptidoglycan recognition proteins (PGRPs) constitute a recently characterized family of pattern-recognition molecules that are conserved from insects to humans and are implicated in mammalian innate immunity. Here we report the isolation, characterization, cDNA cloning, and antimicrobial activities of a bovine PGRP ortholog termed bovine oligosaccharide-binding protein (bOBP). Milligram quantities of bOBP were purified from peripheral leukocytes, thus allowing for the characterization of the disulfide array and for determining the in vitro antimicrobial activities of the native protein. Of the tissues analyzed, bOBP mRNA was detected only in bone marrow where the protein is synthesized as a 190 amino acid precursor. The mature 169 amino acid protein is stored in the cytoplasmic granules of neutrophils and eosinophils but is absent from lymphocytes, monocytes, and platelets. bOBP was microbicidal for Gram-positive and Gram-negative bacteria and yeast at low micromolar concentrations. The finding that bOBP was microbicidal for organisms in which peptidoglycan is absent (Cryptococcus neoformans) or buried (Salmonella typhimurium) indicates that previous conclusions about the specificity of peptidoglycan recognition proteins must be reevaluated and suggests that other envelope components may mediate the antimicrobial action of PGRP family members.
Assuntos
Antibacterianos/farmacologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Plaquetas/metabolismo , Northern Blotting , Western Blotting , Células da Medula Óssea/metabolismo , Bovinos , Células Cultivadas , Clonagem Molecular , Cisteína/química , Citoplasma/metabolismo , DNA Complementar/metabolismo , Dissulfetos/química , Relação Dose-Resposta a Droga , Eosinófilos/metabolismo , Imuno-Histoquímica , Leucócitos/metabolismo , Dados de Sequência Molecular , Monócitos/metabolismo , Neutrófilos/metabolismo , Oligossacarídeos/metabolismo , Biossíntese Peptídica , Peptidoglicano/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
P18 (KWKLFKKIPKFLHLAKKF-NH(2)) is an alpha-helical antimicrobial peptide designed from a cecropin A-magainin 2 hybrid. In this study, P18 was found to show strong antimicrobial activity against several antibiotic-resistant bacterial and fungal strains. Both the salt resistance on antimicrobial activity and the synergistic effect with clinically used antibiotic agents are critical factors in developing effective peptide antibiotic drugs. For this reason, we investigated the salt resistance of P18 to antagonism by NaCl, CaCl(2), and MgCl(2) on antimicrobial activity and the synergistic effect of P18 with vancomycin against vancomycin-resistant Enterococcus faecium (VREF). Compared to magainin 2, P18 showed strong resistance on antimicrobial activity against bacterial strains and C. albicans under high NaCl concentrations of 100-200 mM. In addition, P18 displayed much greater salt resistance on antibacterial activity against Gram-negative bacteria at the physiological or elevated concentrations of CaCl(2) and MgCl(2) than magainin 2. Furthermore, the combination study revealed that P18 has a relatively effective synergistic effect with vancomycin against VREF. Thus, these results support that P18 may prove to be a salt-resistant antibiotic peptide potentially useful in the treatment of cystic fibrosis patients as well as a valuable adjuvant for antimicrobial chemotherapy.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos/farmacologia , Sais/farmacologia , Vancomicina/farmacologia , Proteínas de Xenopus , Adjuvantes Farmacêuticos/farmacologia , Antivirais/farmacologia , Cloreto de Cálcio/farmacologia , Candida albicans/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Sinergismo Farmacológico , Enterococcus faecalis/metabolismo , Magaininas , Cloreto de Magnésio/farmacologia , Biossíntese Peptídica , Ligação Proteica , Estrutura Secundária de Proteína , Cloreto de Sódio/farmacologiaRESUMO
On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.
Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Arginina/química , Leucina/química , Peptídeos/química , Peptídeos/farmacologia , Adjuvantes Imunológicos/isolamento & purificação , Sequência de Aminoácidos , Animais , Arginina/isolamento & purificação , Cálcio/metabolismo , Cromatografia em Agarose , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cisteína/química , Bases de Dados Factuais , Relação Dose-Resposta a Droga , Histamina/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Leucina/isolamento & purificação , Mastócitos/metabolismo , Meliteno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/metabolismo , Biossíntese Peptídica , Peptídeos/isolamento & purificação , Ligação Proteica , Conformação Proteica , Rana pipiens , Análise de Sequência de Proteína , Pele/química , Temperatura , Fatores de TempoRESUMO
Natural products have served as an important source of medicinal compounds and pharmaceutical leads over the last century. Within the last 10 years, significant interest has developed in applying combinatorial chemistry techniques to the study of natural products and their biological activities. In this review, we examine several representative efforts wherein natural product skeletons have been constructed or immobilized on solid support and subsequently derivatized, giving rise to analog libraries useful in understanding the structure-activity relationships of the parent natural product. Issues such as target selection, library design, linker development, automation, and library characterization are addressed.
Assuntos
Técnicas de Química Combinatória , Diterpenos , Epotilonas , Biossíntese Peptídica , Peptídeos/síntese química , Alcaloides/síntese química , Antibacterianos/síntese química , Antineoplásicos/síntese química , Antioxidantes/síntese química , Cicloparafinas/síntese química , Estrogênios não Esteroides/síntese química , Macrolídeos/síntese química , Modelos Químicos , Biblioteca de Peptídeos , Selênio/química , Relação Estrutura-Atividade , Vancomicina/síntese química , Zearalenona/síntese químicaRESUMO
omega-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha(1B-d)) and peripheral (alpha(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta(3) in Xenopus oocytes. However, the potency of CVID and MVIIA increased when alpha(1B-d) and alpha(1B-b) were expressed in the absence of rat beta(3), an effect most pronounced for CVID at alpha(1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. (1)H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.
Assuntos
Canais de Cálcio/metabolismo , Neurônios/metabolismo , ômega-Conotoxinas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Ligação de Hidrogênio , Íons , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Biossíntese Peptídica , Peptídeos/química , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Caramujos , Fatores de Tempo , Ducto Deferente/metabolismo , Xenopus laevis , ômega-Conotoxinas/química , ômega-Conotoxinas/genética , ômega-Conotoxinas/farmacologiaRESUMO
Many types of physical, spectroscopic, and biological studies of proteins and other macromolecules are facilitated by the incorporation of reporter groups. In many cases these are single atom substitutes, for example isotopes (13C for C), or light (F for H) and heavy (Se for S) atom homologs. In some circumstances the incorporation of two different labels in the same molecule would be greatly desirable. Commonly used protein engineering methods for incorporating them can rarely cope with differential double labeling, and have other limitations such as universal, non-specific, or random incorporation. Although de novo peptide synthesis has the power to achieve highly specific labeling, the difficulties inherent in creating long sequences lead us to propose protein semisynthesis as the most practical approach. By ligating combinations of natural and labeled synthetic fragments to reform holoproteins, we can overcome any of the limitations discussed. Using cytochrome c as a model protein we show that two reporter atoms, selenium and bromine, can be simultaneously and site-specifically incorporated without significant consequences to structure and (or) function. This capability opens up the prospect of advances in a number of areas in structural biology.
Assuntos
Bromo/metabolismo , Grupo dos Citocromos c/metabolismo , Diagnóstico por Imagem , Selênio/metabolismo , Animais , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Grupo dos Citocromos c/química , Cavalos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/metabolismo , Biossíntese Peptídica , Selenometionina/química , Relação Estrutura-AtividadeRESUMO
In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.
Assuntos
Ácido Aspártico/química , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Proteínas Quinases Dependentes de AMP Cíclico/química , Humanos , Modelos Químicos , Dados de Sequência Molecular , Biossíntese Peptídica , Pró-Colágeno/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.