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1.
Nat Commun ; 10(1): 1402, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926793

RESUMO

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
2.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889875

RESUMO

A novel dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractional precipitation and gel filtration chromatography. The effects of temperature, pH and some metal ions and chemicals on dextranase activity were investigated. Subsequently, the dextranase was used to produce dextran with specific molecular mass. Weight-average molecular mass (Mw) and the ratio of weight-average molecular mass/number-average molecular mass, or polydispersity index (Mw/Mn), of dextran were measured by multiple-angle laser light scattering (MALS) combined with gel permeation chromatography (GPC). The dextranase was purified to 16.09-fold concentration; the recovery rate was 29.17%; and the specific activity reached 350.29 U/mg. Mw of the dextranase was 66 kDa, which is similar to dextranase obtained from other Penicillium species reported previously. The highest activity was observed at 55 °C and a pH of 5.0. This dextranase was identified as an endodextranase, which specifically degraded the α-1,6 glucosidic bonds of dextran. According to metal ion dependency tests, Li⁺, Na⁺ and Fe2+ were observed to effectively improve the enzymatic activity. In particular, Li⁺ could improve the activity to 116.28%. Furthermore, the dextranase was efficient at degrading dextran and the degradation rate can be well controlled by the dextranase activity, substrate concentration and reaction time. Thus, our results demonstrate the high potential of this dextranase from Penicillium cyclopium CICC-4022 as an efficient enzyme to produce specific clinical dextrans.


Assuntos
Dextranase/isolamento & purificação , Dextranase/metabolismo , Penicillium/enzimologia , Cromatografia em Gel , Dextranos/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Íons , Cinética , Metais/farmacologia , Padrões de Referência , Espalhamento de Radiação , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente , Fatores de Tempo
3.
Cell Commun Signal ; 17(1): 14, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30786936

RESUMO

BACKGROUND: Glucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation. RESULTS: Our kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism. CONCLUSIONS: Our results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Cinética , Camundongos , Mitocôndrias/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fatores de Tempo
4.
Int J Biol Macromol ; 126: 1167-1176, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625353

RESUMO

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin­I an interesting model for further understanding of the action of laticifer fluids in plant defense.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteólise , Thevetia/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Filogenia , Inibidores de Proteases/farmacologia , Substâncias Redutoras/química , Análise de Sequência de Proteína , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
5.
Mol Biol Cell ; 30(4): 453-466, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30586322

RESUMO

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.


Assuntos
Aminoaciltransferases/metabolismo , Arginina/metabolismo , Movimento Celular , Dictyostelium/citologia , Dictyostelium/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Actinas/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Dictyostelium/efeitos dos fármacos , Mutação/genética , Fenótipo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/efeitos dos fármacos
6.
Int J Biol Macromol ; 126: 653-661, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30594625

RESUMO

Flavonoids are widely distributed phytochemicals in vegetables, fruits and medicinal plants. Recent studies demonstrate that some natural flavonoids are potent inhibitors of the human UDP-glucuronosyltransferase 1A1 (UGT1A1), a key enzyme in detoxification of endogenous harmful compounds such as bilirubin. In this study, the inhibitory effects of 56 natural and synthetic flavonoids on UGT1A1 were assayed, while the structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated. The results demonstrated that the C-3 and C-7 hydroxyl groups on the flavone skeleton would enhance UGT1A1 inhibition, while flavonoid glycosides displayed weaker inhibitory effects than their corresponding aglycones. Further investigation on inhibition kinetics of two strong flavonoid-type UGT1A1 inhibitors, acacetin and kaempferol, yielded interesting results. Both flavonoids were competitive inhibitors against UGT1A1-mediated NHPN-O-glucuronidation, but were mixed and competitive inhibitors toward UGT1A1-mediated NCHN-O-glucuronidation, respectively. Furthermore, docking simulations showed that the binding areas of NHPN, kaempferol and acacetin on UGT1A1 were highly overlapping, and convergence with the binding area of bilirubin within UGT1A1. In summary, detailed structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated carefully and the findings shed new light on the interactions between flavonoids and UGT1A1, and will contribute considerably to the development of flavonoid-type drugs without strong UGT1A1 inhibition.


Assuntos
Flavonoides/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Domínio Catalítico , Flavonas/química , Flavonas/farmacologia , Flavonoides/química , Corantes Fluorescentes/metabolismo , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Humanos , Concentração Inibidora 50 , Quempferóis/química , Quempferóis/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Especificidade por Substrato/efeitos dos fármacos
7.
Aquat Toxicol ; 204: 9-18, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30170209

RESUMO

The major hazard of arsenic in living organisms is increasingly being recognized. Marine mollusks are apt to accumulate high levels of arsenic, but knowledge related to arsenic detoxification in marine mollusks is still less than sufficient. In this study, arsenic bioaccumulation as well as the role of glutathione S-transferase omega (GSTΩ) in the process of detoxification were investigated in the Ruditapes philippinarum clam after waterborne exposure to As(III) or As(V) for 30 days. The results showed that the gills accumulated significantly higher arsenic levels than the digestive glands. Arsenobetaine (AsB) and dimethylarsenate (DMA) accounted for most of the arsenic found, and monomethylarsonate (MMA) can be quickly metabolized. A subcellular distribution analysis showed that most arsenic was in biologically detoxified metal fractions (including metal-rich granules and metallothionein-like proteins), indicating their important roles in protecting cells from arsenic toxicity. The relative mRNA expressions of two genes encoding GSTΩ were up-regulated after arsenic exposure, and the transcriptional responses were more sensitive to As(III) than As(V). The recombinant GSTΩs exhibited high activities at optimal conditions, especially at 37 °C and pH 4-5, with an As(V) concentration of 60 mM. Furthermore, the genes encoding GSTΩ significantly enhance the arsenite tolerance but not the arsenate tolerance of E. coli AW3110 (DE3) (ΔarsRBC). It can be deduced from these results that GSTΩs play an important role in arsenic detoxification in R. philippinarum.


Assuntos
Arsênico/metabolismo , Bivalves/enzimologia , Glutationa Transferase/metabolismo , Animais , Arseniato Redutases/genética , Arseniato Redutases/metabolismo , Arseniatos/toxicidade , Arsênico/toxicidade , Bivalves/citologia , Bivalves/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Concentração de Íons de Hidrogênio , Inativação Metabólica/efeitos dos fármacos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de Proteína , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente , Distribuição Tecidual/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade
8.
Biomed Pharmacother ; 105: 526-532, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29885636

RESUMO

The state of enzymes in the human body determines the normal physiology or pathology, so all the six classes of enzymes are crucial. Proteases, the hydrolases, can be of several types based on the nucleophilic amino acid or the metal cofactor needed for their activity. Cathepsins are proteases with serine, cysteine, or aspartic acid residues as the nucleophiles, which are vital for digestion, coagulation, immune response, adipogenesis, hormone liberation, peptide synthesis, among a litany of other functions. But inflammatory state radically affects their normal roles. Released from the lysosomes, they degrade extracellular matrix proteins such as collagen and elastin, mediating parasite infection, autoimmune diseases, tumor metastasis, cardiovascular issues, and neural degeneration, among other health hazards. Over the years, the different types and isoforms of cathepsin, their optimal pH and functions have been studied, yet much information is still elusive. By taming and harnessing cathepsins, by inhibitors and judicious lifestyle, a gamut of malignancies can be resolved. This review discusses these aspects, which can be of clinical relevance.


Assuntos
Catepsinas/metabolismo , Animais , Catepsinas/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Inibidores de Proteases/farmacologia , Especificidade por Substrato/efeitos dos fármacos
9.
Biomed Res Int ; 2018: 9634349, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29850593

RESUMO

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two enzymes sensitive to various chemical compounds having ability to bind to crucial parts of these enzymes. Boldine is a natural alkaloid and it was mentioned in some older works that it can inhibit some kinds of AChE. We reinvestigated this effect on AChE and also on BChE using acetyl (butyryl) thiocholine and Ellman's reagents as standard substances for spectrophotometric assay. We found out IC50 of AChE equal to 372 µmol/l and a similar level to BChE, 321 µmol/l. We conclude our experiment by a finding that boldine is cholinesterase inhibitor; however we report significantly weaker inhibition than that suggested in literature. Likewise, we tried to investigate the mechanism of inhibition and completed it with in silico study. Potential toxic effect on cholinesterases in real conditions is also discussed.


Assuntos
Aporfinas/farmacologia , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Metabolismo Secundário , Acetilcolinesterase/metabolismo , Aporfinas/química , Butirilcolinesterase/química , Inibidores da Colinesterase/química , Células HEK293 , Humanos , Modelos Moleculares , Especificidade por Substrato/efeitos dos fármacos
10.
J Biotechnol ; 280: 31-37, 2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-29860055

RESUMO

Feruloylated acylglycerols (FAG) can be used as antioxidants and UV absorbing ingredients in food and cosmetics. In this work, FAG was prepared by the lipase-catalyzed transesterification of glyceryl monoferulate (GMF) with different acyl donors using ionic liquids (ILs) as reaction solvents. The effect of different imidazolium ILs (BF4-, PF6- and TF2N-) and acyl donors (monoacylglycerols and diacylglycerols) on the transesterification and lipase selectivity for FAG formation were compared. The effect of reaction parameters (temperature, enzyme concentration, substrates ratio and time) on the reaction were also studied. The results showed that FAG preparation can be enhanced using monoolein (MO) and distearin (DS) as acyl donors. High transesterification activity and excellent lipase selectivity for lipophilic FAG formation were achieved using [C18MIM]PF6 as reaction solvent. The activation energy to form the lipophilic FAG by transesterification using MO as an acyl donor was 37.2 kJ/mol, which was lower than that of DS (92.9 kJ/mol). The activation energy to form the hydrophilic glyceryl diferulate by the esterification of GMF with feruloyl formed by the hydrolysis of another GMF (21.5 kJ/mol) using MO as an acyl donor was lower than that of DS (61.9 kJ/mol).


Assuntos
Biocatálise , Ácidos Cumáricos/metabolismo , Glicerídeos/biossíntese , Líquidos Iônicos/farmacologia , Lipase/metabolismo , Biocatálise/efeitos dos fármacos , Diglicerídeos/metabolismo , Esterificação , Cinética , Monoglicerídeos/metabolismo , Solventes , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente , Fatores de Tempo
11.
PLoS One ; 13(5): e0197476, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29775464

RESUMO

Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 5'-GCTGGCGC-3' (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3'-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.


Assuntos
Exodesoxirribonuclease V/metabolismo , Pseudomonas/enzimologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regiões Antárticas , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Bacteriano/metabolismo , Exodesoxirribonuclease V/isolamento & purificação , Hidrólise , Magnésio/farmacologia , Proteínas Mutantes/metabolismo , Mutação/genética , Plasmídeos/metabolismo , Pseudomonas syringae/enzimologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
12.
Int J Biol Macromol ; 116: 451-462, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29730006

RESUMO

Chikungunya virus (CHIKV), a mosquito-borne pathogenic alphavirus is a growing public health threat. No vaccines or antiviral drug is currently available in the market for chikungunya treatment. nsP2pro, the viral cysteine protease, carries out an essential function of nonstructural polyprotein processing and forms four nonstructural proteins (nsPs) that makes the replication complex, hence constitute a promising drug target. In this study, crystal structure of nsP2pro has been determined at 2.59 Å, which reveals that the protein consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Structural comparison of CHIKV nsP2pro with structures of other alphavirus nsP2 advances that the substrate binding cleft is present at the interface of two subdomains. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This loop contains His548, the catalytic residue, and Trp549 and Asn547, the residues predicted to bind substrate. Interestingly, mutation of Asn547 leads to three-fold increase in Km confirming that Asn547 plays important role in substrate binding and recognition. This study presents the detailed molecular analysis and signifies the substrate specificity residues of CHIKV nsP2pro, which will be beneficial for structure-based drug design and optimization of CHIKV protease inhibitors.


Assuntos
Vírus Chikungunya/química , Cisteína Proteases/química , Proteínas não Estruturais Virais/química , Antivirais/farmacologia , Domínio Catalítico/efeitos dos fármacos , Vírus Chikungunya/efeitos dos fármacos , Cristalografia por Raios X/métodos , Desenho de Drogas , Inibidores de Proteases/farmacologia , Especificidade por Substrato/efeitos dos fármacos
13.
Biomed Pharmacother ; 99: 511-522, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29665654

RESUMO

OBJECTIVE: Cancer stem-like cells (CSLCs) are considered a root of tumorigenicity and resistance. However, their identification remains challenging. The use of the side population (SP) assay as a credible marker of CSLCs remains controversial. The SP assay relies on the elevated activity of ABC transporters that, in turn, can be modulated by hypericin (HYP), a photosensitizer and bioactive compound of St. John's Wort (Hypericum perforatum), a popular over-the-counter antidepressant. Here we aimed to comprehensively characterize the SP phenotype of cancer cells and to determine the impact of HYP on these cells. METHODS: Flow cytometry and sorting-based assays were employed, including CD24-, CD44-, CD133-, and ALDH-positivity, clonogenicity, 3D-forming ability, ABC transporter expression and activity, and intracellular accumulation of HYP/Hoechst 33342. The tumorigenic ability of SP, nonSP, and HYP-treated cells was verified by xenotransplantation into immunodeficient mice. RESULTS: The SP phenotype was associated with elevated expression of several investigated transporters and more intensive growth in non-adherent conditions but not with higher clonogenicity, tumorigenicity or ALDH-positivity. Despite stimulated BCRP level and MRP1 activity, HYP reversibly decreased the SP proportion, presumably via competitive inhibition of BCRP. HYP-selected SP cells acquired additional traits of resistance and extensively eliminated HYP. CONCLUSIONS: Our results suggest that SP is not an unequivocal CSLC-marker. However, SP could play an important role in modulating HYP-treatment and serve as a negative predictive tool for HYP-based therapies. Moreover, the use of supplements containing HYP by cancer patients should be carefully considered, due to its proposed effect on drug efflux and complex impact on tumor cells, which have not yet been sufficiently characterized.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Perileno/análogos & derivados , Células da Side Population/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Células Clonais , Humanos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Perileno/farmacologia , Fenótipo , Células da Side Population/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Especificidade por Substrato/efeitos dos fármacos , Análise de Sobrevida
14.
Mar Drugs ; 16(4)2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29642383

RESUMO

Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7). In this study, the kinetic constants Vmax (maximum reaction rate) and Km (Michaelis constant) of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the ß-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm) by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM), and poly-guluronic acid (polyG), resulting in the production of oligosaccharides with a degree of polymerization (DP) of 2-9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.


Assuntos
Organismos Aquáticos/enzimologia , Proteínas de Bactérias/química , Polissacarídeo-Liase/química , Cloreto de Sódio/farmacologia , Vibrio/enzimologia , Alginatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Endopeptidases , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
15.
Can J Physiol Pharmacol ; 96(8): 719-727, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29430946

RESUMO

The endothelium is crucial for the maintenance of vascular tone by releasing several vasoactive substances, including nitric oxide (NO). Systemic mean arterial pressure is primarily regulated by the resistance vasculature, which has been shown to exhibit increased vascular reactivity, and decreased vasorelaxation during hypertension. Here, we aimed to determine the mechanism for mesenteric artery vasorelaxation of the stroke-prone spontaneously hypertensive rat (SHRSP). We hypothesized that endothelial NO synthase (eNOS) is upregulated in SHRSP vessels, increasing NO production to compensate for the endothelial dysfunction. Concentration-response curves to acetylcholine (ACh) were performed in second-order mesenteric arteries; we observed decreased relaxation responses to ACh (maximum effect elicited by the agonist) as compared with Wistar-Kyoto (WKY) controls. Vessels from SHRSP incubated with Nω-nitro-l-arginine methyl ester and (or) indomethacin exhibited decreased ACh-mediated relaxation, suggesting a primary role for NO-dependent relaxation. Vessels from SHRSP exhibited a significantly decreased relaxation response with inducible NO synthase (iNOS) inhibition, as compared with WKY vessels. Western blot analysis showed increased total phosphorylated NF-κB, and phosphorylated and total eNOS in SHRSP vessels. Overall, these data suggest a compensatory role for NO by increased eNOS activation. Moreover, we believe that iNOS, although increasing NO bioavailability to compensate for decreased relaxation, leads to a cycle of further endothelial dysfunction in SHRSP mesenteric arteries.


Assuntos
Artérias Mesentéricas/patologia , Artérias Mesentéricas/fisiopatologia , Óxido Nítrico/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia , Vasodilatação , Acetilcolina/farmacologia , Animais , Arginase/antagonistas & inibidores , Arginase/metabolismo , Arginina/farmacologia , Pressão Sanguínea , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Masculino , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Endogâmicos SHR , Especificidade por Substrato/efeitos dos fármacos , Sístole , Vasodilatação/efeitos dos fármacos
16.
Int J Biol Macromol ; 112: 767-774, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29427680

RESUMO

d-Allulose 3-epimerase (DAEase) catalyzes the epimerization between d-fructose and d-allulose. We had PCR-cloned and overexpressed the gene encoding Agrobacterium sp. ATCC 31749 DAEase (AsDAEase) in Escherichia coli. A high yield of active AsDAEase, 35,300U/L or 1350U/g of wet cells, was acquired with isopropyl ß-d-1-thiogalactopyranoside induction at 20°C for 20h. Although only six residues including residue 234 located in tetrameric interface are different between AsDAEase and A. tumefaciens DAEase (AtDAEase), the specific activity of purified AsDAEase is much larger than that of AtDAEase. The optimal pHs and optimal temperatures of the purified recombinant AsDAEase are 7.5-8.0 and 55-60°C, respectively. The half-life of the enzyme is 267min at 55°C in the presence of 0.1mM Co2+, and the equilibrium ratio between d-allulose and d-fructose is 30:70 at 55°C. Besides characterizing AsDAEase, mutation N234D was constructed to assess its influence on activity. The specific activity of the purified N234D AsDAEase is only 25.5% of wild-type's activity, suggesting residue N234 is an important interfacial residue which substantially affects enzyme activity. The high specific activity and high expression yield of AsDAEase suggest its prospect to be applied in d-allulose production.


Assuntos
Agrobacterium tumefaciens/enzimologia , Aminoácidos/metabolismo , Carboidratos Epimerases/metabolismo , Proteínas Recombinantes/metabolismo , Carboidratos Epimerases/química , Carboidratos Epimerases/isolamento & purificação , Cobalto/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Frutose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , Homologia Estrutural de Proteína , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
17.
Aquat Toxicol ; 197: 109-121, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29462762

RESUMO

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.


Assuntos
Acetilcolinesterase/metabolismo , Crassostrea/enzimologia , Monitoramento Ambiental , Estuários , Temperatura Ambiente , Animais , Biocatálise/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Praguicidas/toxicidade , Especificidade por Substrato/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade
18.
Molecules ; 23(2)2018 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-29462992

RESUMO

Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. ß-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for ß-propeller phytase functions could be of great benefit to biotechnology.


Assuntos
6-Fitase/genética , Bactérias/enzimologia , Metagenômica , 6-Fitase/antagonistas & inibidores , 6-Fitase/química , Sequência de Aminoácidos , Bactérias/genética , Meio Ambiente , Estabilidade Enzimática/efeitos dos fármacos , Biblioteca Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Íons , Metais/farmacologia , Modelos Moleculares , Filogenia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia Estrutural de Proteína , Especificidade por Substrato/efeitos dos fármacos , Temperatura Ambiente
19.
Eur J Pharm Sci ; 114: 364-371, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29292018

RESUMO

The objective of the current study was to characterize and evaluate the functional importance of the Nucleoside Transporters (NTs) in the cornea of the rabbits. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for the molecular characterization of the NTs. Their functionality was evaluated using two substrates, ribavirin and cytarabine. Dipyridamole was used as a blocker for the study. All the treatments were given topically. Molecular characterization of NTs revealed presence of ent1, ent2, ent3 and cnt3 in the cornea. The concentration vs time profile for cytarabine in Aqueous Humor (AH) exhibited a statistically significant (p<0.05) drop at 1h with blocker pretreatment. The mean AUC0-2 between the treatments was also differing in a significant (p<0.05) manner. The concentration vs time profile for ribavirin in AH also showed a significant (p<0.05) decrease in its concentration at 1h with blocker pretreatment. Dipyridamole was able to block ribavirin's entry with as low as 40nM concentration while complete blockade was achieved at 8mM and above. When cytarabine and ribavirin were co-administered, ribavirin at a concentration of 6.5mM significantly inhibited (p<0.05) the transcorneal permeation of cytarabine up to 80%. To conclude, this study showed the presence and functional importance of NTs in the transcorneal uptake of nucleoside substrates. This study further revealed the presence of concentration dependent competitive inhibition among substrates for their transcorneal permeation.


Assuntos
Córnea/efeitos dos fármacos , Córnea/metabolismo , Proteínas de Transporte de Nucleosídeos/administração & dosagem , Proteínas de Transporte de Nucleosídeos/metabolismo , Administração Oftálmica , Administração Tópica , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , Humor Aquoso/efeitos dos fármacos , Humor Aquoso/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Dipiridamol/administração & dosagem , Dipiridamol/metabolismo , Feminino , Masculino , Permeabilidade , Coelhos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia
20.
Xenobiotica ; 48(10): 1037-1049, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28945155

RESUMO

1. Penciclovir, ganciclovir, creatinine, para-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7. 2. For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS. 3. Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC50 of 17, 22, 23, 24, 35 and 586 µM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC50 = 27 µM; >90% inhibition) by plated human hepatocytes (PHH). 4. It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.


Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Aciclovir/análogos & derivados , Aciclovir/farmacologia , Adulto , Estrona/análogos & derivados , Estrona/metabolismo , Feminino , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cetoprofeno/farmacologia , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Peptídeos/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Transfecção
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