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1.
J Sci Food Agric ; 100(1): 315-324, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525262

RESUMO

BACKGROUND: In order to utilize tilapia skin gelatin hydrolysate protein, which is normally discarded as industrial waste in the process of fish manufacture, we study the in vivo and in vitro angiotensin-I-converting enzyme (ACE) inhibitory activity of the peptide Leu-Ser-Gly-Tyr-Gly-Pro (LSGYGP). The aim was to provide a pharmacological basis of the development of minimal side effects of ACE inhibitors by comparative analysis with captopril in molecular docking. RESULTS: This peptide from protein-rich wastes showed excellent ACE inhibitory activity (IC50  = 2.577 µmol L-1 ) and exhibited a mixed noncompetitive inhibitory pattern with Lineweaver-Burk plots. Furthermore, LSGYGP and captopril groups both showed significant decreases in blood pressure after 6 h and maintained good digestive stability over 4 h. Molecular bond interactions differentiate competitive captopril upon hydrogen bond interactions and Zn(II) interaction. The C-terminal Pro generates three interactions (hydrogen bonds, hydrophilic interactions and Van der Waals interactions) in the peptide and effectively interacts with the S1 and S2 pockets of ACE. CONCLUSION: LSGYGP, with an IC50 value of 2.577 µmol L-1 , has an antihypertensive effect in spontaneously hypertensive rats. Through comparison with captopril, this study revealed that LSGYGP may be a potential food-derived ACE inhibitory peptide and could act as a functional food ingredient to prevent hypertension. © 2019 Society of Chemical Industry.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Captopril/química , Hipertensão/tratamento farmacológico , Peptídeos/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Captopril/administração & dosagem , Ciclídeos , Digestão , Proteínas de Peixes/química , Trato Gastrointestinal/metabolismo , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Cinética , Masculino , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Ratos , Ratos Endogâmicos SHR
2.
J Sci Food Agric ; 100(1): 308-314, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525267

RESUMO

BACKGROUND: Peanut is among the most common of food allergies, and one of its allergens is Ara h 2. A previous study revealed that this allergen was recognized by serum immunoglobulin E (IgE) in over 90% of a peanut-allergic patient population. Enzymatic cross-linking is a popular processing method used to tailor food functionality, such as antigenicity. RESULT: The cross-linking reactions of Ara h 2 were catalyzed by polyphenol oxidase (PPO), and the relevant reaction sites were identified using mass spectrometry and StavroX software. Two pairs of intramolecular cross-linking peptides and two intermolecular cross-linking peptides were found. Intramolecular cross-linking was speculated to occur between ARG131 (amino acids 116-131) and TYR65 (amino acids 63-80) and between TYR60 (amino acids 56-62) and ARG92 (amino acids 92-102); the intermolecular cross-linking sites were ARG31 with TYR84 or TYR89 and TYR65 or TYR72 with ARG92 or ARG102 . Three out of four cross-linking peptides were found in α-helices, and destruction of this secondary structure resulted in a loose tertiary structure. Although seven linear allergen epitopes were involved in cross-linking, the IgE binding capacity of protein changed slightly, while its sensitization potential decreased in mouse model. CONCLUSION: Exploring the structural change of Ara h 2 after cross-linking is beneficial in further understanding the influence of structure on sensitization. This result indicated the future possibility of precision processing on structure of proteins to improve their properties. © 2019 Society of Chemical Industry.


Assuntos
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/imunologia , Catecol Oxidase/química , Sequência de Aminoácidos , Arachis/química , Arachis/imunologia , Biocatálise , Epitopos/química , Epitopos/imunologia , Manipulação de Alimentos , Humanos , Imunoglobulina E/imunologia , Hipersensibilidade a Amendoim/imunologia , Estrutura Secundária de Proteína
3.
Gene ; 728: 144287, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31843359

RESUMO

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-binding proteins that play crucial roles in invertebrate immunity. In this study, a novel C-type lectin gene (ScCTL-1) was identified in razor clam Sinonovacula constricta. The ScCTL-1 gene, consisting of four C-type carbohydrate recognition domains (CRDs) with an N-terminal signal peptide and a C-terminal transmembrane region. The gene is widely expressed in almost all tissues, with the highest expression in the hepatopancreas. To explore the functional characteristics of this structurally novel gene, tests of binding specificity, agglutinating activity, and phagocytic promoting activity were included in this study. Bacterial stimulation up-regulated ScCTL-1 expression in hemocytes. The binding activity of rScCTL-1 to bacteria was tested in vitro, and bacterial agglutination was observed under the same conditions. Ca2+ was essential for carbohydrate binding. Additionally, rScCTL-1 promoted the phagocytic activity of hemocytes to varying degrees against different bacteria, unlike the classical opsonin. These results suggest ScCTL-1 is a classical immune-related C-type lectin possessing unique immune-related properties.


Assuntos
Bivalves/genética , Bivalves/imunologia , Hepatopâncreas/metabolismo , Lectinas Tipo C/genética , Fagocitose , Staphylococcus aureus/fisiologia , Vibrio/fisiologia , Aglutinação , Sequência de Aminoácidos , Animais , Bivalves/microbiologia , Carboidratos/química , Filogenia , Domínios Proteicos , Homologia de Sequência , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
4.
J Agric Food Chem ; 67(46): 12918-12926, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31668066

RESUMO

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.


Assuntos
Braquiúros/enzimologia , Epitopos/química , Triose-Fosfato Isomerase/imunologia , Sequência de Aminoácidos , Animais , Braquiúros/química , Braquiúros/genética , Braquiúros/imunologia , Reações Cruzadas , Cristalização , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoglobulina E/imunologia , Conformação Molecular , Conformação Proteica , Alinhamento de Sequência , Frutos do Mar/análise , Hipersensibilidade a Frutos do Mar/imunologia , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética
5.
Chem Commun (Camb) ; 55(92): 13864-13867, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31670736

RESUMO

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.


Assuntos
Corynebacterium glutamicum/metabolismo , Heme/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Heme/química , Ligação Proteica , Estrutura Terciária de Proteína
6.
J Chem Phys ; 151(17): 175102, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703505

RESUMO

Proteins are classified into families based on evolutionary relationships and common structure-function characteristics. Availability of large data sets of gene-derived protein sequences drives this classification. Sequence space is exponentially large, making it difficult to characterize family differences. In this work, we show that Machine Learning (ML) methods can be trained to distinguish between protein families. A number of supervised ML algorithms are explored to this end. The most accurate is a Long Short Term Memory (LSTM) classification method that accounts for the sequence context of the amino acids. Sequences for a number of protein families where there are sufficient data to be used in ML are studied. By splitting the data into training and testing sets, we find that this LSTM classifier can be trained to successfully classify the test sequences for all pairs of the families. Also investigated is whether the addition of structural information increases the accuracy of the binary comparisons. It does, but because there is much less available structural than sequence information, the quality of the training degrades. Another variety of LSTM, LSTM_wordGen, a context-dependent word generation algorithm, is used to generate new protein sequences based on seed sequences for the families considered here. Using the original sequences as training data and the generated sequences as test data, the LSTM classification method classifies the generated sequences almost as accurately as the true family members do. Thus, in principle, we have generated new members of these protein families.


Assuntos
Aprendizado de Máquina , Proteínas/química , Proteínas/classificação , Sequência de Aminoácidos , Conformação Proteica
7.
J Agric Food Chem ; 67(45): 12452-12460, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31674183

RESUMO

Increasing cases of infections by foodborne pathogenic bacteria resulted in a great demand to find safe and novel antimicrobial compounds that can be used in the food industry. The isolation and application of antimicrobial peptides including lipopeptides has been increasing tremendously in the past years. In this study, a new bacterial strain called Brevibacillus laterosporus fmb70 (fmb70) was isolated and exhibited strong antimicrobial activities against Gram-positive, Gram-negative bacteria, and fungi. Two major antimicrobial components produced by fmb70 were respectively identified as lipopeptide: brevibacillin V (MW: 1570.12 Da) and brevibacillin (MW: 1583.75 Da), of which brevibacillin V was a new compound. Both of them consisted of 13 amino acids and C6 fatty acyl (FA) chain. Brevibacillin V and brevibacillin showed significant antimicrobial activities against most foodborne pathogenic bacteria and phytopathogenic fungi. They stayed activity at 100 °C and remained 50% of their antimicrobial activities at pH 3 for 22 h. Hemolytic activities of them were lower than 8%. They effectively eliminated the S. aureus GIM 1.142 and L. monocytogenes ATCC 21633 in skim milk. In conclusion, the Brevibacillus laterosporus fmb70 and its major antimicrobial components has remarkable potentials in the food industry.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Brevibacillus/química , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Leite/microbiologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Brevibacillus/metabolismo , Bovinos , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Lipopeptídeos/metabolismo , Testes de Sensibilidade Microbiana , Mapeamento de Peptídeos
8.
J Agric Food Chem ; 67(46): 12816-12823, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31675231

RESUMO

Microbial degradation plays a major role in the dissipation of pendimethalin, and nitroreduction is an initial and detoxicating step. Previously, a pendimethalin nitroreductase, PNR, was identified in Bacillus subtilis Y3. Here, another pendimethalin nitroreductase from strain Y3, LNR, was identified. LNR shares only 40% identity with PNR and reduces the aromatic ring C-6 nitro group of pendimethalin and both nitro groups of trifluralin, butralin, and oryzalin. The catalytic activities against the four dinitroanilines were much higher for LNR than for PNR. lnr deletion significantly reduced the pendimethalin-reduction activity (60% activity loss), while pnr deletion led to only 30% activity loss, indicating that both LNR and PNR were involved in pendimethalin nitroreduction in strain Y3; however, LNR played the major role. This study facilitates the elucidation of pendimethalin catabolism and provides degrading enzyme resources for the removal of dinitroaniline herbicide residues in environment and agricultural products.


Assuntos
Compostos de Anilina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Herbicidas/metabolismo , Nitrorredutases/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Bacillus subtilis/classificação , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Herbicidas/química , Nitrorredutases/química , Nitrorredutases/genética , Filogenia , Alinhamento de Sequência
10.
Photochem Photobiol Sci ; 18(11): 2657-2660, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624823

RESUMO

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.


Assuntos
Proteínas de Bactérias/metabolismo , Methylobacterium/metabolismo , Oxigênio Singlete/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Polarização de Fluorescência , Mutagênese Sítio-Dirigida , Oxigênio/química , Ligação Proteica , Teoria Quântica , Alinhamento de Sequência , Ureia/química
11.
J Agric Food Chem ; 67(44): 12219-12227, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31613626

RESUMO

Quantification, using an accurate analytical approach, of capsinoids and capsaicinoids was performed on three chili pepper (Capsicum spp.) genotypes: "Chiltepín", "Tampiqueño 74", and "Bhut Jolokia" at various stages of fruit development. The accumulation of capsinoids, in all these peppers started between 10 to 20 days post-anthesis (dpa), increased and reached the highest capsinoid amount at 40 dpa, and then decreased until 60 dpa. Conversely, capsaicinoids could already be determined at 10 dpa in "Bhut Jolokia" and their accumulation pattern was different from that of the capsinoids in this genotype. The capsiate/dihydrocapsiate ratio presented a higher variation between genotypes and developmental stages than the capsaicin/dihydrocapsaicin ratio. Capsinoid ratios (4-24%) and Pun1/pAMT genotyping were determined. These results provide information on the progress of the accumulation of capsinoids in the aforementioned pungent and superhot cultivars and could support future breeding studies toward the understanding of the factors affecting their accumulation.


Assuntos
Capsaicina/análogos & derivados , Capsaicina/metabolismo , Capsicum/genética , Capsicum/metabolismo , Aromatizantes/metabolismo , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/genética , Alelos , Sequência de Aminoácidos , Capsaicina/análise , Capsicum/química , Capsicum/crescimento & desenvolvimento , Aromatizantes/análise , Frutas/química , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Genótipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
12.
Plant Mol Biol ; 101(4-5): 507-516, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31617145

RESUMO

KEY MESSAGE: MMDH2 gene negatively regulates Cd tolerance by modulating reactive oxygen species (ROS) levels and the ROS-mediated signaling, thus, affecting the expression of PDR8. The molecular mechanism by which plants respond to stress caused by cadmium (Cd), one of the most toxic heavy metals to plants, is not well understood. Here, we show that MMDH2, a gene encoding mitochondrial malate dehydrogenase, is involved in Cd stress tolerance in Arabidopsis. The expression of MMDH2 was repressed by Cd stress. The mmdh2 knockdown mutants showed enhanced Cd tolerance, while the MMDH2-overexpressing lines were sensitive to Cd. Under normal and Cd stress conditions, lower H2O2 levels were detected in mmdh2 mutant plants than in wild-type plants. In contrast, higher H2O2 levels were found in MMDH2-overexpressing lines, and they were negatively correlated with malondialdehyde levels. In addition, the expression of the PDR8, a gene encoding a Cd efflux pump, increased and decreased in the mmdh2 mutant and MMDH2-overexpressing lines, in association with lower and higher Cd concentrations, respectively. These results suggest that the MMDH2 gene negatively regulates Cd tolerance by modulating reactive oxygen species (ROS) levels and the ROS-mediated signaling, thus, affecting the expression of PDR8.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Cádmio/metabolismo , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
13.
Protein Pept Lett ; 26(10): 768-775, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618171

RESUMO

INTRODUCTION: Metallothioneins (MTs) are members of a family of low molecular weight and cysteine-rich proteins that are involved in heavy metal homeostasis and detoxification in living organisms. Plants have multiple MT types that are generally divided into four subgroups according to the arrangement of Cys residues. METHODS: In the present study the E. coli cells which heterologously express four different rice MT (OsMT) isoforms were analyzed for the accumulation of two forms of chromium, Cr3+ and Cr6+. RESULTS: The results show that the transgenic bacteria were more tolerant than control cells when they were grown up in the medium comprising Cr(NO3)3.9H2O or Na2CrO4. The cells expressing OsMT1, OsMT2, OsMT3 and OsMT4 give rise to 6.5-, 2.7-, 5.5- and 2.1-fold improvements on the accumulation capacity for Cr3+ and 9-, 3-, 5- and 3- fold Cr6+ respectively compared with comparison to the control strain. Furthermore, the purified recombinant GST-OsMTs were tested for their binding ability to Cr+3 and Cr+6 in vitro. DISCUSSION: The data show that the recombinant GST-OsMT1 and GST-OsMT2 were able to bind both Cr3+ and Cr6+, in vitro. However, their binding strength was low with respect to previous tested divalent ions like Cd2+.


Assuntos
Cromo/química , Metalotioneína/química , Oryza/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Cátions/química , Cisteína/química , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Metalotioneína/genética , Proteínas de Plantas/genética , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/genética
14.
Protein Pept Lett ; 26(10): 792-797, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618172

RESUMO

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.


Assuntos
Proteína Axina/química , Proteína Axina/genética , Proteínas Desgrenhadas/química , Domínios Proteicos/genética , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X/métodos , Proteínas Desgrenhadas/genética , Escherichia coli , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Via de Sinalização Wnt
15.
Chemistry ; 25(64): 14572-14582, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31599485

RESUMO

Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.


Assuntos
Nisina/análogos & derivados , Peptídeos/síntese química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Ligações de Hidrogênio , Nisina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
16.
Int J Nanomedicine ; 14: 7795-7808, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576124

RESUMO

Background: Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. Methods: In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. Results: The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. Conclusion: The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.


Assuntos
Sistemas de Liberação de Medicamentos , MicroRNAs/metabolismo , Peptídeos/química , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia , Sequência de Aminoácidos , Animais , Adesão Celular , Gotículas Lipídicas/metabolismo , Camundongos , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/química , Transfecção
17.
J Agric Food Chem ; 67(42): 11758-11768, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31577438

RESUMO

Patulin contamination not only is a menace to human health but also causes serious environmental problems worldwide due to the synthetic fungicides that are used to control it. This study focused on investigating the patulin degradation mechanism in Pichia caribbica at the molecular level. According to the results, P. caribbica (2 × 106 cells/mL) was able to degrade patulin from 20 µg/mL to an undetectable level in 72 h. The RNA-seq data showed patulin-induced oxidative stress and responses in P. caribbica. The deletion of PcCRG1 led to a significant decrease in patulin degradation by P. caribbica, whereas the overexpression of PcCRG1 accelerated the degradation of patulin. The study identified that PcCRG1 protein had the ability to degrade patulin in vitro. Overall, we demonstrated that the patulin degradation process in P. caribbica was more than one way; PcCRG1 was an S-adenosylmethionine-dependent methyltransferase and played an important role in the patulin degradation process in P. caribbica.


Assuntos
Proteínas Fúngicas/metabolismo , Fungicidas Industriais/metabolismo , Metiltransferases/metabolismo , Patulina/metabolismo , Pichia/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Metiltransferases/química , Metiltransferases/genética , Pichia/enzimologia , Pichia/genética , Alinhamento de Sequência
18.
Int J Nanomedicine ; 14: 7053-7064, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564865

RESUMO

Background: Food allergy (FA) is a significant public health problem. The therapeutic efficacy for FA is unsatisfactory currently. The breakdown of intestinal immune tolerance is associated with the pathogenesis of FA. Therefore, it is of great significance to develop novel therapeutic methods to restore immune tolerance in treating FA. Methods: We proposed an oral administration strategy to treat FA by co-delivering food allergen epitope fragment (peptide: IK) and adjuvant R848 (TLR7 ligand) in the mPEG-PDLLA nanoparticles (PPLA-IK/R848 NPs). The generation of tolerogenic dendritic cells (DCs) and regulatory T cells (Tregs) induced by PPLA-IK/R848 NPs were evaluated in vitro and in vivo. The therapeutic effects of PPLA-IK/R848 NPs were also assessed in an OVA-induced FA model. Results: PPLA-IK/R848 NPs could efficiently deliver IK to DCs to drive DCs into the tolerogenic phenotypes and promote the differentiation of Tregs in vitro and in vivo, significantly inhibited FA responses through the recovery of intestinal immune tolerance. Conclusion: Oral administration of PPLA-IK/R848 NPs could efficiently deliver IK and R848 to intestinal DCs and stimulate DCs into allergen tolerogenic phenotype. These tolerogenic DCs could promote the differentiation of Tregs, which significantly protected mice from food allergic responses. This study provided an efficient formulation to alleviate FA through the recovery of immune tolerance.


Assuntos
Alérgenos/imunologia , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos , Epitopos/imunologia , Hipersensibilidade Alimentar/tratamento farmacológico , Hipersensibilidade Alimentar/imunologia , Imidazóis/administração & dosagem , Imidazóis/uso terapêutico , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Imidazóis/química , Imidazóis/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Peptídeos/química , Poliésteres/química , Polietilenoglicóis/química , Linfócitos T Reguladores/efeitos dos fármacos
19.
Phys Chem Chem Phys ; 21(43): 24147-24164, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31657381

RESUMO

Understanding the selectivity mechanisms of inhibitors towards highly similar proteins is extremely important work on the way to a new drug. Here, we aim to reveal the selectivity mechanisms of type I 1/2 kinase inhibitors towards p21-activated kinase (PAK4) and mitogen-activated protein kinase kinase kinase 14 (MAP3K14, NIK). PAK4, belonging to the serine/threonine protein kinases, is involved in cell signaling pathways and controls cellular functions and has received attention as an attractive drug target. The high sequence identity between PAK4 and NIK makes it challenging to design selective PAK4 inhibitors. In this work, computational methods including protein comparison, molecular docking, QM/MM, molecular dynamics simulations, and density functional theory (DFT) calculation were employed to explore the binding mechanisms of selective inhibitors against NIK and PAK4. The simulation results revealed the crucial factors accounting for selective inhibition of PAK4 over NIK, including different protein-ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. This study will shed light on understanding the selectivity mechanisms of PAK4 and NIK inhibitors.


Assuntos
Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Quinases Ativadas por p21/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Teoria da Densidade Funcional , Humanos , Ligações de Hidrogênio , Camundongos , Análise de Componente Principal , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Quinases Ativadas por p21/metabolismo
20.
Phys Chem Chem Phys ; 21(39): 22103-22112, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31570909

RESUMO

Type III phosphatidylinositol 4 kinases (PI4KIIIs) are essential enzymes that are related to the replication of multiple RNA viruses. Understanding the interaction mechanisms of molecular compounds with the alpha and beta isoforms of PI4KIII (PI4KIIIα and PI4KIIIß) is of significance in the development of inhibitors that can bind to these two enzymes selectively. In this work, molecular dynamics (MD) simulations and binding free energy calculations were combined to investigate the binding modes of seven selected compounds to PI4KIIIα and PI4KIIIß. Analyses based on MD trajectories provide detailed interaction mechanisms of these compounds with PI4KIIIα and PI4KIIIß at the atomic level, and indicate that the selectivity of these compounds is mainly due to the structural difference of the binding pockets. It is expected that the detailed binding information found in this study can provide useful help for the structure-based design of selective inhibitors toward PI4KIIIα and PI4KIIIß.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Domínio Catalítico , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Termodinâmica
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