Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 16.116
Filtrar
1.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Chem Commun (Camb) ; 57(67): 8352-8355, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34337637

RESUMO

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-ß-d-glucose tetraacetate) and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-ß-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 µM for TGTA-RRE and 33 ± 2 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.


Assuntos
Inibidores Enzimáticos/farmacologia , Ferro/farmacologia , Óxidos de Nitrogênio/farmacologia , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Ferro/química , Modelos Moleculares , Estrutura Molecular , Óxidos de Nitrogênio/química , SARS-CoV-2/enzimologia
3.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445215

RESUMO

Zea mays L. is one of the most produced crops, and there are still parts of the world where maize is the basic staple food. To improve agriculture, mankind always looks for new, better methods of growing crops, especially in the current changing climatic conditions. Cold atmospheric pressure plasma (CAPP) has already showed its potential to enhance the culturing of crops, but it still needs more research for safe implementation into agriculture. In this work, it was shown that short CAPP treatment of maize grains had a positive effect on the vitality of grains and young seedlings, which may be connected to stimulation of antioxidant and lytic enzyme activities by short CAPP treatment. However, the prolonged treatment had a negative impact on the germination, growth, and production indexes. CAPP treatment caused the increased expression of genes for heat shock proteins HSP101 and HSP70 in the first two days after sowing. Using comet assay it was observed that shorter treatment times (30-120 s) did not cause DNA damage. Surface diagnostics of plasma-treated grains showed that plasma increases the hydrophilicity of the surface but does not damage the chemical bonds on the surface.


Assuntos
Grão Comestível/crescimento & desenvolvimento , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Gases em Plasma/farmacologia , Fatores de Transcrição/metabolismo , Zea mays/crescimento & desenvolvimento , Pressão Atmosférica
4.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445562

RESUMO

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway; however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3high therapeutic-refractory cancer.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Epidérmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Complexo do Signalossomo COP9/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeo Hidrolases/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
5.
Nutrients ; 13(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34444848

RESUMO

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.


Assuntos
Proteínas de Bactérias/metabolismo , Flavonoides/metabolismo , Microbioma Gastrointestinal/fisiologia , Fenômenos Fisiológicos da Nutrição/fisiologia , Simulação por Computador , Equol/metabolismo , Genoma Bacteriano , Humanos , Isoflavonas/metabolismo , Metagenoma , Peptídeo Hidrolases/metabolismo , Proteólise
6.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206113

RESUMO

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.


Assuntos
Bronquiectasia/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Bronquiectasia/enzimologia , Bronquiectasia/genética , Bronquiectasia/patologia , Humanos , Elastase de Leucócito , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Serina Proteases/genética , Serina Proteases/metabolismo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
7.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198602

RESUMO

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.


Assuntos
Ensaios Enzimáticos/métodos , Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Desnaturação Proteica , Especificidade por Substrato , Temperatura
8.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281200

RESUMO

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved ß-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.


Assuntos
Endopeptidases/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Peptidoglicano/metabolismo , Prófagos/genética , Prófagos/metabolismo , Ligação Proteica , Staphylococcus/metabolismo , Staphylococcus aureus/metabolismo , Especificidade por Substrato
9.
J Enzyme Inhib Med Chem ; 36(1): 1646-1650, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34289752

RESUMO

The chemical structure of PF-07321332, the first orally available Covid-19 clinical candidate, has recently been revealed by Pfizer. No information has been provided about the interaction pattern between PF-07321332 and its biomolecular counterpart, the SARS-CoV-2 main protease (Mpro). In the present work, we exploited Supervised Molecular Dynamics (SuMD) simulations to elucidate the key features that characterise the interaction between this drug candidate and the protease, emphasising similarities and differences with other structurally related inhibitors such as Boceprevir and PF-07304814. The structural insights provided by SuMD will hopefully be able to inspire the rational discovery of other potent and selective protease inhibitors.


Assuntos
Antivirais/química , Lactamas/química , Leucina/química , Simulação de Dinâmica Molecular , Nitrilas/química , Prolina/química , Inibidores de Proteases/química , Antivirais/farmacologia , Humanos , Lactamas/farmacologia , Leucina/farmacologia , Ligantes , Nitrilas/farmacologia , Peptídeo Hidrolases/metabolismo , Prolina/farmacologia , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Software
10.
Methods Mol Biol ; 2341: 9-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264455

RESUMO

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.


Assuntos
N-Acetil-Muramil-L-Alanina Amidase/análise , Peptídeo Hidrolases/análise , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Staphylococcus aureus/enzimologia
11.
J Enzyme Inhib Med Chem ; 36(1): 1267-1281, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34210221

RESUMO

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.


Assuntos
Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Inibidores de Proteases/farmacologia , Tannerella forsythia/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/química , Tannerella forsythia/isolamento & purificação , Temperatura
12.
Chem Commun (Camb) ; 57(63): 7778-7781, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34263896

RESUMO

Cyclative cleavage of an amine-carbamate self-immolative spacer to deliver a hydroxyl cargo was inhibited by spacer derivatisation with a phosphate monoester handle. This trifunctional spacer was installed in a model anticancer prodrug that showed fast drug release only when incubated with both a protease and a phosphatase enzyme.


Assuntos
Antineoplásicos/metabolismo , Peptídeo Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Pró-Fármacos/metabolismo , Antineoplásicos/química , Liberação Controlada de Fármacos , Estrutura Molecular , Pró-Fármacos/química
13.
Biomed Pharmacother ; 139: 111611, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243597

RESUMO

Trichomonas vaginalis is an amitochondriate protozoan and the agent of human trichomoniasis, the most prevalent non-viral sexually transmitted infection (STI) in the world. In this study we showed that 2,4-diamine-quinazoline derivative compound (PH100) kills T. vaginalis. PH100 showed activity against fresh clinical and American Type Culture Collection (ATCC) T. vaginalis isolates with no cytotoxicity against cells (HMVI, 3T3-C1 and VERO) and erythrocytes. In addition, PH100 showed synergistic action with metronidazole, indicating that these compounds act by different mechanisms. When investigating the mechanism of action of PH100 to ATCC 30236, apoptosis-like characteristics were observed, such as phosphatidylserine exposure, membrane alterations, and modulation of gene expression and activity of peptidases related to apoptosis. The apoptosis-like cell death features were not observed for the fresh clinical isolate treated with PH100 revealing distinct profiles. Our data revealed the heterogeneity among T. vaginalis isolates and contribute with the understanding of mechanisms of cell death in pathogenic eukaryotic organisms without mitochondria.


Assuntos
Diaminas/farmacologia , Parasitos/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Quinazolinas/farmacologia , Vaginite por Trichomonas/tratamento farmacológico , Trichomonas vaginalis/efeitos dos fármacos , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Metronidazol/farmacologia , Camundongos , Vaginite por Trichomonas/parasitologia , Células Vero
14.
J Cell Sci ; 134(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34313310

RESUMO

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA, also known as AURKA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupts the deSUMOylation of AurA, leading to increased kinase activity and abnormalities in spindle assembly and chromosome segregation, which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render spatiotemporal control on its kinase activity in mitosis. This article has an associated First Person interview with the first author of the paper.


Assuntos
Aurora Quinase A , Peptídeo Hidrolases , Aurora Quinase A/genética , Cisteína Endopeptidases/metabolismo , Humanos , Mitose , Peptídeo Hidrolases/metabolismo , Fosforilação , Fuso Acromático/genética , Fuso Acromático/metabolismo
15.
Fish Shellfish Immunol ; 116: 52-60, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34216786

RESUMO

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.


Assuntos
Ácidos e Sais Biliares/farmacologia , Suplementos Nutricionais , Linguados , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Fosfatase Alcalina/imunologia , Amilases/metabolismo , Animais , Complemento C3/imunologia , Dieta/veterinária , Proteínas de Peixes/metabolismo , Linguados/genética , Linguados/imunologia , Linguados/metabolismo , Linguados/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina M/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lipase/metabolismo , Muramidase/imunologia , Oxirredutases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Junções Íntimas/genética
16.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066609

RESUMO

Pseudomonas aeruginosa (P. aeruginosa), one of the dangerous multidrug resistance pathogens, orchestrates virulence factors production through quorum sensing (QS). Since the exploration of QS inhibitors, targeting virulence to circumvent bacterial pathogenesis without causing significant growth inhibition is a promising approach to treat P. aeruginosa infections. The present study has evaluated the anti-QS and anti-infective activity of epigallocatechin-3-gallate (EGCG), a bioactive ingredient of the traditional green tea, against P. aeruginosa. EGCG showed significant inhibitory effects on the development of biofilm, protease, elastase activity, swimming, and swarming motility, which was positively related to the production of C4-AHL. The expression of QS-related and QS-regulated virulence factors genes was also evaluated. Quantitative PCR analysis showed that EGCG significantly reduced the expression of las, rhl, and PQS genes and was highly correlated with the alterations of C4-AHL production. In-vivo experiments demonstrated that EGCG treatment reduced P. aeruginosa pathogenicity in Caenorhabditis elegans (C. elegans). EGCG increased the survival of C. elegans by 23.25%, 30.04%, and 36.35% in a dose-dependent manner. The findings of this study strongly suggest that EGCG could be a potential candidate for QS inhibition as an anti-virulence compound against bacterial infection.


Assuntos
Biofilmes/crescimento & desenvolvimento , Catequina/análogos & derivados , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Acil-Butirolactonas/metabolismo , Animais , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Catequina/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/biossíntese , Testes de Sensibilidade Microbiana , Movimento , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/biossíntese , Percepção de Quorum/genética
17.
Int J Nanomedicine ; 16: 3755-3773, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34103914

RESUMO

Purpose: Acinetobacter baumannii antibiotic resistant infections in high-risk patients are a great challenge for researchers and clinicians worldwide. In an effort to achieve potent bactericidal outcomes, a novel chitosan-mastoparan nanoconstruct (Mast-Cs NC) was designed and assessed for its therapeutic potential through in silico, in vitro and in vivo experimentation against clinical multidrug-resistant (MDR) A. baumannii. Methods: Optimized 3D structures of mastoparan and chitosan were coupled computationally through an ionic cross-linker to generate a circular ring of chitosan encasing mastoparan. The complex was assessed for interactions and stability through molecular dynamic simulation (MDS). Binding pocket analysis was used to assess the protease-peptide interface. Mast-Cs NC were prepared by the ionic gelation method. Mast-Cs NC were evaluated in vitro and in vivo for their therapeutic efficacy against drug-resistant clinical A. baumannii. Results: MDS for 100 ns showed stable bonds between chitosan and mastoparan; the first at chitosan oxygen atom-46 and mastoparan isoleucine carbon atom with a distance of 2.77 Å, and the second between oxygen atom-23 and mastoparan lysine nitrogen atom with a distance of 2.80 Å, and binding energies of -3.6 and -7.4 kcal/mol, respectively. Mast-Cs complexes approximately 156 nm in size, with +54.9 mV zeta potential and 22.63% loading capacity, offered >90% encapsulation efficiency and were found to be geometrically incompatible with binding pockets of various proteases. The MIC90 of Mast-Cs NC was significantly lower than that of chitosan (4 vs 512 µg/mL, respectively, p<0.05), with noticeable bacterial damage upon morphological analysis. In a BALB/c mouse sepsis model, a significant reduction in bacterial colony count in the Mast-Cs treated group was observed compared with chitosan and mastoparan alone (p<0.005). Mast-Cs maintained good biocompatibility and cytocompatibility. Conclusion: Novel mastoparan-loaded chitosan nanoconstructs signify a successful strategy for achieving a synergistic bactericidal effect and higher therapeutic efficacy against MDR clinical A. baumannii isolates. The Mast-Cs nano-drug delivery system could work as an alternative promising treatment option against MDR A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Quitosana/química , Simulação por Computador , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Nanopartículas/química , Venenos de Vespas/farmacologia , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/isolamento & purificação , Adolescente , Adulto , Animais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Nanopartículas/ultraestrutura , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Adulto Jovem
18.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065111

RESUMO

Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco-obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway dysfunction, and impair the processes that could resolve disease. We focus on how proteases regulate the state of mucus at the airway surface, impair mucociliary clearance and ultimately, promote mucostasis. We discuss how, in parallel, proteases are able to promote an inflammatory environment in the airways by mediating proinflammatory signalling, compromising host defence mechanisms and perpetuating their own proteolytic activity causing structural lung damage. Finally, we discuss some possible reasons for the clinical inefficacy of protease inhibitors to date and propose that, especially in a combination therapy approach, proteases represent attractive therapeutic targets for muco-obstructive lung diseases.


Assuntos
Imunidade nas Mucosas , Pneumopatias/etiologia , Pneumopatias/metabolismo , Muco/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Doença Crônica , Cílios/imunologia , Cílios/metabolismo , Suscetibilidade a Doenças , Humanos , Transporte de Íons , Pneumopatias/diagnóstico , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais
19.
J Infect Dis ; 224(Supplement_1): S1-S21, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111271

RESUMO

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/virologia , Desenvolvimento de Medicamentos , Humanos , National Institutes of Health (U.S.) , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Estados Unidos , Replicação Viral/efeitos dos fármacos
20.
Molecules ; 26(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071573

RESUMO

Mass spectrometry analyses carried out on mass spectrometers equipped with soft ionization sources demonstrated their utility in the assessment of the formation of noncovalent complexes and the localization of the binding sites. Direct analyses by mass spectrometry of the noncovalent complex formed in acidic and mildly acidic environments by amyloid beta (1-40) peptide and oleuropein have been previously described, and, in several studies, the absorption, metabolism, excretion, and the implications in the prevention and therapy of Alzheimer's disease of oleuropein have been investigated. Our paper presents modifications of the method previously employed for noncovalent complex observation, namely, the amyloid beta (1-40) pretreatment, followed by an increase in the pH and replacement of the chemical environment from ammonium acetate to ammonium bicarbonate. The formation of noncovalent complexes with one or two molecules of oleuropein was detected in all chemical solutions used, and the amyloid beta (17-28) binding site was identified via proteolytic experiments using trypsin prior to and after noncovalent complex formation. Our results highlight the importance of further studies on the effect of oleuropein against amyloid beta aggregation.


Assuntos
Peptídeos beta-Amiloides/química , Glucosídeos Iridoides/química , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Acetatos/química , Doença de Alzheimer/metabolismo , Bicarbonatos/química , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Proteólise , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Tripsina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...