RESUMO
Vicilins are related to cowpea seed resistance toward Callosobruchus maculatus due to their ability to bind to chitinous structures lining larval midgut. However, this binding mechanism is not fully understood. Here, we identified chitin binding sites and investigated how in vitro and in silico chemical modifications interfere with vicilin chitin binding and insect toxicity. In vitro assays showed that unmodified vicilin strongly binds to chitin matrices, mainly with acetylated chitin. Chemical modifications of specific amino acids (tryptophan, lysine, tyrosine), as well as glutaraldehyde cross-linking, decreased the evaluated parameters. In silico analyses identified at least one chitin binding site in vicilin monomer, the region between Arg208 and Lys216, which bears the sequence REGIRELMK and forms an α helix, exposed in the 3D structure. In silico modifications of Lys223 (acetylated at its terminal nitrogen) and Trp316 (iodinated to 7-iodine-L-tryptophan or oxidized to ß-oxy-indolylalanine) decreased vicilin chitin binding affinity. Glucose, sucrose, and N-acetylglucosamine also interfered with vicilin chitin binding affinity.
Assuntos
Quitina/metabolismo , Besouros/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Quitina/química , Besouros/química , Besouros/efeitos dos fármacos , Simulação por Computador , Larva/química , Larva/efeitos dos fármacos , Larva/metabolismo , Ligação Proteica , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Vigna/química , Vigna/genética , Vigna/metabolismoRESUMO
Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding ß-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of ß-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to ß-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of ß-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.
Assuntos
Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes/genética , Vigna/genética , Animais , Sítios de Ligação , Quitina/química , Quitina/genética , Clonagem Molecular , Besouros/patogenicidade , DNA Complementar/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/química , Ligação Proteica , Proteínas de Armazenamento de Sementes/química , Sementes/química , Sementes/genética , Vigna/crescimento & desenvolvimentoRESUMO
The objective of this research was to identify and characterize the encoded peptides present in nut storage proteins of Carya illinoinensis. It was found, through in silico prediction, proteomic analysis, and MS spectrometry, that bioactive peptides were mainly found in albumin and glutelin fractions. Glutelin was the major fraction with ~53% of the nut storage proteins containing at least 21 peptides with different putative biological activities, including antihypertensives, antioxidants, immunomodulators, protease inhibitors, and inhibitors of cell cycle progression in cancer cells. Data showed that using 50 µg/mL tryptic digests of enriched peptides obtained from nut glutelins is able to induce up to 19% of apoptosis in both HeLa and CasKi cervical cancer cells. To our knowledge, this is the first report that shows the potential value of the nut-encoded peptides to be considered as adjuvants in cancer therapies.
Assuntos
Antioxidantes/química , Carya/química , Peptídeos/química , Proteínas de Armazenamento de Sementes/genética , Antioxidantes/isolamento & purificação , Carya/genética , Glutens/química , Glutens/genética , Nozes/química , Nozes/genética , Peptídeos/genética , Peptídeos/isolamento & purificação , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Proteômica , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Sementes/químicaRESUMO
Seeds from Cucurbitaceae plants (squashes, pumpkins, melons, etc.) have been used both as protein-rich food ingredients and nutraceutical agents by many indigenous cultures for millennia. However, relatively little is known about the bioactive components (e.g., peptides) of the Cucurbitaceae seed proteins (CSP) and their specific effects on human health. Therefore, this paper aims to provide a comprehensive review of latest research on bioactive and functional properties of CSP isolates and hydrolysates. Enzymatic hydrolysis can introduce a series of changes to the CSP structure and improve its bioactive and functional properties, including the enhanced protein solubility over a wide range of pH values. Small-sized peptides in CSP hydrolysates seem to enhance their bioactive properties but adversely affect their functional properties. Therefore, medium degrees of hydrolysis seem to benefit the overall improvement of bioactive and functional properties of CSP hydrolysates. Among the reported bioactive properties of CSP isolates and hydrolysates, their antioxidant, antihypertensive, and antihyperglycaemic activities stand out. Therefore, they could potentially substitute synthetic antioxidants and drugs which might have adverse secondary effects on human health. CSP isolates and hydrolysates could also be implemented as functional food ingredients, thanks to their favorable amino acid composition and good emulsifying and foaming properties.
Assuntos
Antioxidantes/química , Cucurbitaceae/química , Peptídeos/química , Hidrolisados de Proteína/química , Proteínas de Armazenamento de Sementes/química , Animais , HumanosRESUMO
Plants have evolved sophisticated embryo defences by kinetically-stable non-digestible storage proteins that lower the nutritional value of seeds, a strategy that have not been reported in animals. To further understand antinutritive defences in animals, we analysed PmPV1, massively accumulated in the eggs of the gastropod Pomacea maculata, focusing on how its structure and structural stability features affected its capacity to withstand passage through predator guts. The native protein withstands >50 min boiling and resists the denaturing detergent sodium dodecyl sulphate (SDS), indicating an unusually high structural stability (i.e., kinetic stability). PmPV1 is highly resistant to in vitro proteinase digestion and displays structural stability between pH 2.0-12.0 and 25-85 °C. Furthermore, PmPV1 withstands in vitro and mice digestion and is recovered unchanged in faeces, supporting an antinutritive defensive function. Subunit sequence similarities suggest a common origin and tolerance to mutations. This is the first known animal genus that, like plant seeds, lowers the nutritional value of eggs by kinetically-stable non-digestible storage proteins that survive the gut of predators unaffected. The selective pressure of the harsh gastrointestinal environment would have favoured their appearance, extending by convergent evolution the presence of plant-like hyperstable antinutritive proteins to unattended reproductive stages in animals.
Assuntos
Trato Gastrointestinal/metabolismo , Plantas/genética , Estabilidade Proteica , Proteínas de Armazenamento de Sementes/genética , Animais , Ovos/análise , Cinética , Camundongos , Valor Nutritivo/genética , Valor Nutritivo/imunologia , Comportamento Predatório/fisiologia , Conformação Proteica , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/imunologia , Sementes/química , Sementes/genética , Caramujos/química , Caramujos/genéticaRESUMO
Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the ß-subunit from ß-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.
Assuntos
Antígenos de Plantas/imunologia , Enterococcus faecalis/metabolismo , Globulinas/imunologia , Proteínas de Armazenamento de Sementes/imunologia , Leite de Soja/metabolismo , Proteínas de Soja/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fermentação , Globulinas/química , Globulinas/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/metabolismo , Leite de Soja/química , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Glycine max/química , Glycine max/imunologia , Glycine max/metabolismo , Glycine max/microbiologiaRESUMO
A new family of small plant peptides was recently described and found to be widespread throughout the Millereae and Heliantheae tribes of the sunflower family Asteraceae. These peptides originate from the post-translational processing of unusual seed-storage albumin genes, and have been termed PawS-derived peptides (PDPs). The prototypic family member is a 14-residue cyclic peptide with potent trypsin inhibitory activity named SunFlower Trypsin Inhibitor (SFTI-1). In this study we present the features of three new PDPs discovered in the seeds of the sunflower species Zinnia haageana by a combination of de novo transcriptomics and liquid chromatography-mass spectrometry. Two-dimensional solution NMR spectroscopy was used to elucidate their structural characteristics. All three Z. haageana peptides have well-defined folds with a head-to-tail cyclized peptide backbone and a single disulfide bond. Although two possess an anti-parallel ß-sheet structure, like SFTI-1, the Z. haageana peptide PDP-21 has a more irregular backbone structure. Despite structural similarities with SFTI-1, PDP-20 was not able to inhibit trypsin, thus the functional roles of these peptides is yet to be discovered. Defining the structural features of the small cyclic peptides found in the sunflower family will be useful for guiding the exploitation of these peptides as scaffolds for grafting and protein engineering applications.
Assuntos
Asteraceae/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Proteínas de Armazenamento de Sementes/química , Peptídeos Cíclicos/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas de Armazenamento de Sementes/isolamento & purificaçãoRESUMO
The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects.
Assuntos
Antifúngicos/farmacologia , Capsicum/química , Peptídeos/farmacologia , Proteínas de Armazenamento de Sementes/farmacologia , Sementes/química , Leveduras/efeitos dos fármacos , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antifúngicos/isolamento & purificação , Cromatografia por Troca Iônica , Inibidores Enzimáticos/farmacologia , Humanos , Insetos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Micologia , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Alinhamento de Sequência , Leveduras/crescimento & desenvolvimento , alfa-Amilases/metabolismoRESUMO
Canary grass is used as traditional food for diabetes and hypertension treatment. The aim of this work is to characterize the biological activity of encrypted peptides released after gastrointestinal digestion of canary seed proteins. Canary peptides showed 43.5% inhibition of dipeptidyl peptidase IV (DPPIV) and 73.5% inhibition of angiotensin-converting enzyme (ACE) activity. An isolated perfused rat heart system was used to evaluate the canary seed vasoactive effect. Nitric oxide (NO), a major vasodilator agent, was evaluated in the venous effluent from isolated perfused rat heart. Canary seed peptides (1 µg/mL) were able to induce the production of NO (12.24 µM) in amounts similar to those induced by captopril (CPT) and bradykinin (BK). These results show that encrypted peptides in canary seed have inhibitory activity against DPPIV and ACE, enzymes that are targets for diabetes and hypertension treatments.
Assuntos
Anti-Hipertensivos/farmacologia , Hipoglicemiantes/farmacologia , Peptídeos/farmacologia , Phalaris/química , Sementes/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Bradicinina , Captopril , Vasos Coronários/efeitos dos fármacos , Digestão , Masculino , Miocárdio/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Ratos , Ratos Wistar , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (µ = 0.2) and acidic pH (<4.1), the recombinant proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.