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1.
Gene ; 766: 145159, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32971186

RESUMO

Considering the relevance of insect α-amylases and natural α-amylase inhibitors present in plants to protect against insect damage, we investigated the effect of white bean and rapeseed protein extracts on digestive α-amylase gene expression of the Colorado potato beetle, Leptinotarsa decemlineata (Say). For this purpose, in vitro and in vivo trials were performed to determine the inhibitory activity of seed proteins on the third and fourth instar larvae. In both trials, the significant inhibitory effect of each extracts on the third and fourth instar larval α-amylase activity and considerable mortality in treatments were observed compared to control trials. In the RT-qPCR, expression ratio demonstrated that the α-amylase gene of two different larval stages grown on both proteins treated leaves had significantly differentiated expression and was up-regulated in third instar larvae and down-regulated in fourth instar larvae compared to control. Results suggest that the hyper-production of α-amylase in third instar larvae is elicited to compensate for the enzyme activity inhibition at an earlier stage and also down-regulation suggests the existence of a negative feedback of plant proteins on the last instar larvae via impaired food intake and digestive α-amylase activity in Colorado potato beetle. Therefore, disruption of the insect's digestive physiology by plant defensive proteins can be considered in the development of innovative controlling methods of this crucial potato pest.


Assuntos
Besouros/efeitos dos fármacos , Besouros/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Larva/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteínas de Plantas/farmacologia , alfa-Amilases/genética , Animais , Digestão/efeitos dos fármacos , Digestão/genética , Regulação da Expressão Gênica/genética , Larva/genética , Folhas de Planta/química , Solanum tuberosum/parasitologia
2.
Chem Biol Interact ; 329: 109223, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32781033

RESUMO

Thromboembolism is a major cause of morbidity and mortality worldwide. Most therapeutic drugs for treating thrombosis can cause hemorrhage and have short half-lives within human blood circulation resulting in a need to discover and develop novel anticoagulants/antithrombotics. EuRP-61 has been isolated from a plant latex (Euphorbia resinifera) and characterized as a serine protease. In this study, EuRP-61 was able to hydrolyze all chains of human fibrin clots. The enzyme may have long term stability in blood circulation as its fibrinogenolytic activity was not affected by human blood circulating inhibitors such as α2-macroglobulin and antithrombin III. The enzyme may affect the extrinsic, intrinsic or common pathways of the human blood coagulation cascade as evidenced by its prolonged of both prothrombin (PT) and activated partial thromboplastin (APTT) time. Moreover, the enzyme inhibited platelet aggregation via the ADP-receptor pathway. EuRP-61 was not toxic to human red blood cells in the 4 common blood groups (A, B, O and AB) (all Rh+) or human peripheral blood mononuclear cells (hPBMCs). The enzyme may protect human peripheral blood cells from aggregation without destroying them. This study provides evidence that EuRP-61 may have potential as an agent for the treatment of thrombosis.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Euphorbia/enzimologia , Fibrinolíticos/farmacologia , Peptídeo Hidrolases/farmacologia , Proteínas de Plantas/farmacologia , Anticoagulantes/isolamento & purificação , Antitrombina III/antagonistas & inibidores , Antitrombina III/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fibrinolíticos/isolamento & purificação , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , alfa 2-Macroglobulinas Associadas à Gravidez/antagonistas & inibidores , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo
3.
Food Chem ; 333: 127481, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663753

RESUMO

In this study, we tested the exogenous application of phytosulfokine α (PSKα) for delaying the yellowing of broccoli florets during cold storage. Our results showed that the lower yellowing in broccoli florets treated with 150 nM PSKα was probably due to the higher endogenous accumulation of PSKα, leading to the endogenous accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP). Besides, broccoli florets treated with 150 nM PSKα exhibited a higher accumulation of phenols and flavonoids by triggering gene expression and activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). Moreover, the higher expression of L-galactotno-1,4-lactone dehydrogenase (GLDH) gene and the lower expression of ascorbic acid oxidase (AAO) gene in broccoli florets treated with 150 nM PSKα may be the reasons for the higher accumulation of ascorbic acid. In conclusion, the exogenous application of PSKα is a promising strategy in delaying the yellowing and preserving the nutritional quality of broccoli florets during cold storage.


Assuntos
Brassica/efeitos dos fármacos , Brassica/metabolismo , Temperatura Baixa , Qualidade dos Alimentos , Armazenamento de Alimentos , Valor Nutritivo/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Ácido Ascórbico/metabolismo , Fenóis/metabolismo , Pigmentação/efeitos dos fármacos
4.
Cancer Sci ; 111(9): 3184-3194, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32589330

RESUMO

Immunotoxins are Ab-cytotoxin chimeric molecules with mighty cytotoxicity. Programmed cell death 1-ligand 1 (PD-L1), is a transmembrane protein expressed mainly in inflammatory tumor tissues and plays a pivotal role in immune escape and tumor progression. Although PD-L1 immune checkpoint therapy has been successful in some cases, many patients have not benefited enough due to primary/secondary resistance. In order to optimize the therapeutic efficacy of anti-PD-L1 mAb, we used durvalumab as the payload and CUS245C , a type I ribosome-inactivating protein isolated from Cucurbita moschata, as the toxin moiety, to construct PD-L1-specific immunotoxin (named D-CUS245C ) through the engineered cysteine residue. In vitro, D-CUS245C selectively killed PD-L1+ tumor cells. In vivo studies also showed that D-CUS245C had obvious antitumor effect on PD-L1+ human xenograft tumors in nude mice. In conclusion, in the combination of the toxin with mAb, this study developed a new immunotoxin targeting PD-L1, emphasizing a novel and promising treatment strategy and providing a valuable way to optimize cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Imunotoxinas/farmacologia , Proteínas de Plantas , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Imunofenotipagem , Imunotoxinas/química , Camundongos , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Transporte Proteico , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Proc Natl Acad Sci U S A ; 117(27): 16043-16054, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571919

RESUMO

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.


Assuntos
Antifúngicos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Simbiose , Sequência de Aminoácidos , Botrytis/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Cisteína/química , Fusarium/metabolismo , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/microbiologia , Espectroscopia de Ressonância Magnética , Medicago truncatula/microbiologia , Pichia/metabolismo , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
6.
Food Chem ; 331: 127355, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32593042

RESUMO

Ara h1 is a major allergen from peanut. We investigated the effect of covalent conjugation of Ara h1 and dietary polyphenols on allergenicity and functional properties of Ara h1. Enzyme-linked immunosorbent assay revealed that the covalent conjugation of dietary polyphenols significantly reduced the IgE binding capacity of Ara h1. Covalent binding of dietary polyphenols with Ara h1 reduced histamine release by 40% in basophils. The decreased IgE binding capacity of Ara h1 could be ascribed to changes in protein conformation. The IgE epitope of Ara h1 might be blocked by polyphenols at the binding site. Analysis of pepsin digestion of Ara h1-polyphenol conjugates indicated that the covalent binding increased pepsin digestibility and reduced IgE binding capacity. Furthermore, covalent conjugation of Ara h1 with polyphenols decreased denaturation temperature and increased antioxidant activity. Ara h1 conjugated with polyphenols may be a promising approach for reducing the allergenicity of Ara h1.


Assuntos
Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Catequina/análogos & derivados , Ácido Clorogênico/química , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Antígenos de Plantas/farmacologia , Antioxidantes/química , Arachis/química , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Basófilos/metabolismo , Catequina/química , Catequina/imunologia , Catequina/metabolismo , Epitopos/metabolismo , Histamina/metabolismo , Humanos , Imunoglobulina E/metabolismo , Proteínas de Membrana/farmacologia , Proteínas de Plantas/farmacologia , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier
7.
Food Chem ; 328: 127135, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32473490

RESUMO

Watermelon seed, a watermelon processing industry by-product, is a good protein source for the preparation of antioxidant peptides due to its high protein content, low cost, special amino acid composition. Antioxidant hydrolysates obtained from watermelon seed protein (WSP) after slit divergent ultrasound (SDU) treatment were studied. The stepwise multiple linear regression model verified that the reducing power of watermelon seed protein hydrolysates (WSPHs) is positively related with -SH and ß-turn content of WSP (R2 = 0.931, p < 0.01). Using the degree of hydrolysis (DH) and reducing power as indicators, the WSPHs was prepared under the optimal conditions (ultrasound frequency: 20/28 kHz, time: 60 min, power density: 100 W/L) and divided into three components by ultrafiltration membrane (1 and 5 kDa). Compared with WSPHs and other fractions, WSPHs-I (Mw < 1 kDa) not only significantly protected HepG2 cells from H2O2-induced damage, but also greatly alleviated the liver injury caused by d-galactose in male SD rats.


Assuntos
Antioxidantes/farmacologia , Citrullus/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Enzimas/metabolismo , Galactose/toxicidade , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise , Modelos Lineares , Masculino , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Ratos Sprague-Dawley , Sementes/química , Ultrassom/instrumentação , Ultrassom/métodos
8.
J Food Sci ; 85(6): 1735-1741, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32468582

RESUMO

In this study, the antioxidant activity of mungbean protein hydrolysate (MPH) was systematically investigated. MPH was fractionated by ultrafiltration into two major fractions (MPH-1 <3 kDa, MPH-2 >3 kDa). Fraction MPH-1, which exhibited the highest antioxidant activity, was further fractionated by gel column into three fractions (MPH-1A, MPH-1B, and MPH-1C). The antioxidant activity of the MPH-1B fraction was stronger than that of the other fractions. Eight mungbean peptides (P1-P8) were identified in fraction MPH-1B by UPLC-Q-TOF-MS. Among them, peptides Trp-Gly-Asn (WGN, P2), Ala-Trp (AW, P4), Arg-Gly-Trp-Tyr-Glu (RGWYE, P5), and Gly-Val-Pro-Phe-Trp (GVPFW, P7) had high antioxidant activity. Moreover, these four peptides exerted protective effects against H2 O2 -induced cytotoxicity and regulated the MDA content, CAT activity, and total GSH content in HepG2 cells with specific observation. This study demonstrated the potential of MPH as a source of antioxidant peptides. This provides a scientific basis for the preparation of antioxidant peptides from mungbean protein. PRACTICAL APPLICATION: This study demonstrated the potential of the hydrolysate of mungbean protein as a source of antioxidant peptides and provided a scientific basis for the preparation of antioxidant peptides from mungbean protein.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Proteínas de Plantas/química , Vigna/química , Sequência de Aminoácidos , Animais , Antioxidantes/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Peptídeos/química , Proteínas de Plantas/farmacologia , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia
9.
Arch Insect Biochem Physiol ; 104(3): e21687, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32342573

RESUMO

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant Ki and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å2 . The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.


Assuntos
Mariposas/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Inibidores da Tripsina/farmacologia , Animais , Fabaceae/química , Larva/efeitos dos fármacos , Larva/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mariposas/enzimologia , Mariposas/crescimento & desenvolvimento , Tripsina/metabolismo
10.
Carbohydr Res ; 490: 107903, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32171073

RESUMO

This study intends to investigate the inhibitory potential of different plant derived saccharides on cell migration and adhesion of pancreatic ductal adenocarcinoma (PDAC) cells to microvascular liver endothelium, particularly considering the role of transmembranous galectin-3. PDAC cell lines PancTu1 and Panc1 were characterized by considerable (transmembranous) galectin-3 (Gal3) expression. SiRNA mediated Gal3 knockdown as well as treatment with differentially processed pectins and arabinogalactan-proteins (AGPs) did not impact on cell migration of either PDAC cell line. In contrast, Gal3 knockdown reduced adhesion of PDAC cells to the liver endothelial cell line TMNK-1 being more pronounced in Panc1 cells. Similarly, plant derived substances did not impact cell adhesion of PancTu1 cells while partially hydrolyzed citrus pectin (MCP), pectinase-treated MCP (MCPPec) and partially hydrolized AGP (AGPTFA) clearly diminished adhesive properties of Panc1 cells. MCPPec or AGPTFA could not further intensify the adhesion reducing effect of galectin-3 knockdown, indicating that these plant derived polysaccharides are able to inhibit PDAC cell adhesion to liver endothelial cells in a galectin-3 dependent manner. Overall, these data suggest an inhibitory potential of plant derived processed saccharides which have undergone chemical modification in impairing PDAC cell adhesion to liver endothelium.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Citrus/química , Galectina 3/metabolismo , Mucoproteínas/farmacologia , Neoplasias Pancreáticas/metabolismo , Pectinas/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galectina 3/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Metástase Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas de Plantas/farmacologia
11.
J Biosci Bioeng ; 130(1): 6-13, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32192842

RESUMO

Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αß structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.


Assuntos
Defensinas/química , Defensinas/metabolismo , Oryza/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cristalização , Defensinas/genética , Defensinas/farmacologia , Oryza/química , Oryza/genética , Fosfatidilinositóis/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia
12.
PLoS One ; 15(3): e0230633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32208440

RESUMO

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.


Assuntos
Degranulação Celular/efeitos dos fármacos , Lectinas/farmacologia , Proteínas de Plantas/farmacologia , Animais , Artocarpus/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Interleucina-6/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Mitose/efeitos dos fármacos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ratos
13.
J Food Sci ; 85(4): 1328-1337, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32220144

RESUMO

Naked oat globulin was hydrolyzed by alcalase, flavourzyme, pepsin, and trypsin in sequence. The hydrolysates (NOGH) were purified using gel chromatography, reversed-phase high performance liquid chromatography (RP-HPLC). Finally, fraction D7d with the highest ACE-inhibitory was subjected to liquid chromatography-mass spectrometry analysis and 14 peptides were identified. Of which, peptide SSYYPFK (890.4 Da) was chose to synthesize based on in silico analysis. The SSYYPFK demonstrated high ACE-inhibitory activity (IC50 : 91.82 µM) with competitive inhibition mode, and could effectively (P < 0.05) lower the systolic blood pressure and diastolic pressure of spontaneously hypertensive rats at the concentration of 100 to 150 mg/kg body weight. Molecular docking simulation demonstrated that SSYYPFK could bind with the active site S1 of ACE via short hydrogen bonds. It could remain the ACE-inhibitory activity after simulated gastrointestinal hydrolysis. Moreover, SSYYPFK showed acceptable renin and endothelin-1 suppressing capacity (47.59% and 27.88% at 1.5 mg/mL, respectively). These results indicated that SSYYPFK may have similar antihypertensive mechanism with captopril, and could be develop to natural antihypertensive products. PRACTICAL APPLICATION: One novel ACE-inhibitory peptide SSYYPFK (890.4 Da) was identified from naked oat globulin hydrolysates. It exhibited relatively high renin and intracellular endothelin-1 suppressing capacity, and could effectively (P < 0.05) lower the systolic blood pressure and diastolic pressure of spontaneously hypertensive rats. This peptide could be used as natural and safe nutraceuticals and/or functional ingredients.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Avena/química , Globulinas/química , Proteínas de Plantas/farmacologia , Animais , Anti-Hipertensivos/química , Avena/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Cromatografia de Fase Reversa , Simulação por Computador , Endotelina-1 , Masculino , Simulação de Acoplamento Molecular , Pepsina A/farmacologia , Peptídeos/química , Peptidil Dipeptidase A/metabolismo , Proteínas de Plantas/química , Hidrolisados de Proteína/química , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Renina , Tripsina
14.
BMC Complement Med Ther ; 20(1): 5, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-32020890

RESUMO

BACKGROUND: Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line. METHODS: NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools. RESULTS: Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 µg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range. CONCLUSION: Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nigella sativa/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/farmacologia , Cromatografia em Gel , Humanos , Células MCF-7 , Espectrometria de Massas , Paquistão , Sementes/química
15.
Cryobiology ; 92: 189-196, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31952948

RESUMO

Late Embryogenesis Abundant (LEA) proteins are commonly found in plants and other organisms capable of undergoing severe and reversible dehydration, a phenomenon termed "anhydrobiosis". Here, we have produced a tagged version for three different LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, binds to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein group 3 that targets the mitochondria. Sheep fibroblasts transfected with single or all three LEA proteins were subjected to air drying under controlled conditions. After rehydration, cell viability and functionality of the membrane/mitochondria were assessed. After 4 h of air drying, cells from the un-transfected control group were almost completely nonviable (1% cell alive), while cells expressing LEA proteins showed high viability (more than 30%), with the highest viability (58%) observed in fibroblasts expressing all three LEA proteins. Growth rate was markedly compromised in control cells, while LEA-expressing cells proliferated at a rate comparable to non-air-dried cells. Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming the protection conferred by LEA proteins on these organelles during dehydration stress. This is likely to be an effective strategy when aiming to confer desiccation tolerance to mammalian cells.


Assuntos
Criopreservação/métodos , Crioprotetores/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Animais , Artemia/metabolismo , Células Cultivadas , Desidratação/metabolismo , Dessecação/métodos , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Ovinos , Triticum/metabolismo , Água/metabolismo , Zea mays/metabolismo
16.
Lett Appl Microbiol ; 70(3): 151-158, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31837275

RESUMO

The aim of this study was to evaluate the effects of synthetic peptides on the growth and biofilm formation of Streptococcus mutans and their cytotoxicity on a human keratinocyte cell line. Four pomegranate-derived peptides; Pug-1, Pug-2, Pug-3 and Pug-4 were synthesized. Streptococcus mutans (ATCC 25175 and DMST 41283) were used. The antibacterial and anti-adherence activities of the peptides were evaluated. The effect of the peptides on S. mutans glucosyltransferase (Gtf) activity was measured. The peptides' cytotoxicity was evaluated on a HaCaT cell line using an MTT assay. The bactericidal penetration depth of Pug-1 into the biofilm was investigated using confocal laser scanning microscopy (CLSM). Among the four peptides, Pug-1 (200 µg ml-1 ) exhibited antibacterial activity on S. mutans (DMST 41283). All peptides demonstrated anti-adherence activity. Streptococcus mutans Gtf activity was significantly inhibited by Pug-1. Interestingly, Pug-1 peptide was not cytotoxic to the HaCaT cell line. Pug-1 (100-200 µg ml-1 ) exhibited bactericidal penetration at approximately 50-100% of the biofilm depth. Therefore, Pug-1 might be a candidate of anticariogenic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data highlighted that pomegranate-derived antimicrobial peptides (AMPs) inhibit Streptococcus mutans adhesion, virulence-associated genes and enzymes. This in vitro study supports the hypothesis that pomegranate AMPs would be noncytotoxic to human keratinocytes, with a potent anticariogenic effect via an anti-adherence mode-of-action. The present study provides evidence for further investigation of these AMPs as therapeutic and preventive agents for dental caries.


Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Plantas/farmacologia , Romã (Fruta)/metabolismo , Streptococcus mutans/crescimento & desenvolvimento , Cárie Dentária/microbiologia , Cárie Dentária/prevenção & controle , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Streptococcus mutans/metabolismo
17.
Proteins ; 88(1): 227-236, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31365155

RESUMO

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Capsicum/química , Bicamadas Lipídicas/metabolismo , Proteínas de Plantas/farmacologia , Tioninas/farmacologia , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Células MCF-7 , Modelos Moleculares , Neoplasias/tratamento farmacológico , Proteínas de Plantas/química , Alinhamento de Sequência , Homologia Estrutural de Proteína , Tioninas/química
18.
J Ethnopharmacol ; 247: 112213, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31562951

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Historical literature and pharmacological studies demonstrate that ginseng, one of the most popular herbal medicines in China, holds potential benefits for Parkinson's disease (PD). AIM OF THE STUDY: Studies in Drosophila melanogaster (Dm) have highlighted mitochondrial dysfunction upon loss of PTEN-induced putative kinase 1 (PINK1) as a central mechanism of PD pathogenesis. Using PINK1B9 mutant Dm, we aimed to explore the therapeutic action of ginseng total protein (GTP) on PD and provide in-depth scientific interpretation about the traditional efficacy of ginseng. MATERIALS AND METHODS: We first used gel chromatography to purify GTP and confirmed its molecular weight by SDS-PAGE. Effects of GTP on PINK1B9 mutants, which were supplied with standard diet from larvae to adult stages, were assayed in flies aged 3-6 (I), 10-15 (II), and 20-25 (III) days. Parkinson-like phenotypes were analyzed by evaluating lifespan, dopaminergic neurons, dopamine levels, and locomotor ability. Mitochondrial function was assessed by evaluating ATP production, respirometry, and mitochondrial DNA. In addition, reactive oxygen species were measured using dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate staining. PD-related oxidative stress was simulated by paraquat and rotenone, and mitochondrial membrane potential was measured using JC-10 reagent. Protein and mRNA expression was detected by Western blot and real-time quantitative reverse transcription polymerase chain reaction, respectively. RESULTS: This study demonstrates for the first time that GTP treatment delays the onset of a Parkinson-like phenotype in PINK1B9 Dm, including prolongation of lifespan and rescue of climbing ability, as well as rescue of the progressive loss of a cluster of dopaminergic neurons in the protocerebral posterior lateral 1 region, which was accompanied by a significant increase of dopamine content in the brain. In addition, GTP notably reduced the penetrance of abnormal wing position, indicating a strong inhibitory effect on indirect flight muscle degeneration. We further showed that GTP could promote maintenance of mitochondrial function and protect mitochondria from PD-associated oxidative stress by activating the mitochondrial unfolded protein response (UPRmt). CONCLUSIONS: GTP protected against mitochondrial dysfunction and neurodegeneration by inducing UPRmt in the Dm PINK1B9 model of PD. Our results suggest that GTP is a promising candidate for PD, and reveal a new mechanism by which ginseng is neuroprotective.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Fármacos Neuroprotetores/farmacologia , Panax/química , Doença de Parkinson/tratamento farmacológico , Proteínas de Plantas/farmacologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas de Plantas/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos
19.
Food Chem ; 308: 125601, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31670190

RESUMO

The aim of this work was to analyse the hypotensive effect of amaranth protein/peptides on spontaneously hypertensive rats (SHR). The mechanism of action of these peptides was studied in vivo and ex vivo. We also tested the effect of protection against gastrointestinal digestion (GID) exerted by an O:W emulsion on the integrity of the antihypertensive peptides. All samples tested produced a decrease in blood pressure (SBP). The animals treated with emulsion (GE) and emulsion + peptide (GE+VIKP) showed the most significant reduction in the SBP (42 ±â€¯2 mmHg and 35 ±â€¯2 mmHg, respectively). The results presented suggest that after GID, a variety of peptides with biological activities were released or were resistant to this process. These peptides play a role in the regulation of the SBP by acting on plasma ACE, plasma renin and the vascular system. These results support the use of amaranth protein/peptides in the elaboration of functional foods for hypertensive individuals.


Assuntos
Amaranthus/química , Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Peptídeos/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Animais , Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Masculino , Peptídeos/uso terapêutico , Proteínas de Plantas/farmacologia , Proteínas de Plantas/uso terapêutico , Ratos , Ratos Endogâmicos SHR
20.
Nanoscale ; 11(48): 23366-23381, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31793603

RESUMO

One of the most important causes of failure in tumour treatment is the development of resistance to therapy. Cancer cells can develop the ability to lose sensitivity to anti-neoplastic drugs during reciprocal crosstalk between cells and their interaction with the tumour microenvironment (TME). Cell-to-cell communication regulates a cascade of interdependent events essential for disease development and progression and can be mediated by several signalling pathways. Exosome-mediated communication is one of the pathways regulating these events. Tumour-derived exosomes (TDE) are believed to have the ability to modulate TMEs and participate in multidrug resistance mechanisms. In this work, we studied the effect of the natural defensin from common bean, PvD1, on the formation of exosomes by breast cancer MCF-7 cells, mainly the modulatory effect it has on the level of CD63 and CD9 tetraspanins. Moreover, we followed the interaction of PvD1 with biological and model membranes of selected composition, by biophysical and imaging techniques. Overall, the results show that PvD1 induces a dual effect on MCF-7 derived exosomes: the peptide attenuates the recruitment of CD63 and CD9 to exosomes intracellularly and binds to the mature exosomes in the extracellular environment. This work uncovers the exosome-mediated anticancer action of PvD1, a potential nutraceutical agent.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Defensinas/farmacologia , Exossomos/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Humanos , Células MCF-7 , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo
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