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1.
Mol Biol (Mosk) ; 53(3): 467-475, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184612

RESUMO

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.


Assuntos
Coriocarcinoma/genética , Coriocarcinoma/patologia , Espectroscopia Dielétrica , Perfilação da Expressão Gênica , Modelos Biológicos , Placenta/citologia , Placenta/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Gravidez
2.
Bull Exp Biol Med ; 166(5): 641-645, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903488

RESUMO

Metastatic cascade is associated with the process of epithelial-mesenchymal transition accompanied by changes in cell proliferation, migration, adhesion, and invasiveness mediated by the insulin-like growth factor (IGF) signal pathway. IGFBP6 protein binds IGF and prevents its interaction with receptors. IGFBP6 gene knockdown through RNA-interference inhibits cell migration and increased the rate of proliferation of breast cancer MDA-MB-231 cells. IGFBP6 knockdown cells are characterized by increased expression of MIR100 and MIRLET7A2 genes encoding hsa-miR-100-3p, hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7a-2-3p miRNA. The target genes of these microRNAs are IGF2, IGF1R, INSR, and CCND1 associated with IGF signaling pathway and proliferative and migratory activity during the metastatic cascade. A significant decrease in the expression of INSR and CCND1 genes was demonstrated by PCR and microarray analysis.


Assuntos
Antígenos CD/metabolismo , Ciclina D1/metabolismo , MicroRNAs/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Antígenos CD/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , Receptor IGF Tipo 1 , Receptor de Insulina/genética , Receptores de Somatomedina/genética
3.
Bull Exp Biol Med ; 166(5): 656-660, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903492

RESUMO

One of actively developing trends in modern pharmacology is the use of the transcriptome analysis for drug repositioning. We have previously detected two molecular markers of relapses in patients with malignant breast tumors: ELOVL5 and IGFBP6. Poor prognosis is associated with low expression of these markers. Here we analyze the effects of simvastatin and a new potential proteasome inhibitor K7174 inducing expression of IGFBP6 and EVOVL5 on the proliferation of breast cancer cells MDA-MB-231 and DU4475. Compound K7174 potentiates the inhibitory effect of simvastatin on the proliferation of DU4475 cells characterized by low expression of ELOVL5-IGFBP6 pair, but not on the proliferation of MDA-MB-231 cells with high expression of these markers.


Assuntos
Neoplasias da Mama/microbiologia , Acetiltransferases/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Combinação de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Elongases de Ácidos Graxos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Recidiva Local de Neoplasia , Sinvastatina/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
4.
Bull Exp Biol Med ; 166(4): 512-516, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30783835

RESUMO

We compared available methods for monitoring the integrity of in vitro models of barrier tissues and studied the possibility of using impedance spectroscopy to solve this problem. It was demonstrated (theoretically and experimentally) that TEER measurements are not sufficiently sensitive to detect small defects in the cell barrier that significantly affect its permeability. For obtaining reliable results, it is necessary to set a sufficiently high threshold TEER, which leads to the loss of many intact samples. At the same time, impedance spectroscopy has all advantages of the classical method of measuring TEER (it is rapid and non-invasive method), while its application in combination with the methods of machine learning allows reliable detection of defects in the cell barrier.


Assuntos
Espectroscopia Dielétrica/métodos , Mucosa Intestinal/metabolismo , Células CACO-2 , Impedância Elétrica , Humanos
5.
Bull Exp Biol Med ; 166(1): 35-38, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30417287

RESUMO

Differentiation of colorectal cancer Caco-2 cells was assessed using Affymetrix Human Gene 1.0 ST arrays and by the main electrical parameters measured by bioimpedance spectroscopy. Transepithelial electrical resistance (TEER) was maximum on day 7, then decreased by day 11, and remained stable. The baseline resistance was maximum on day 4, minimum on day 7, but then gradually increased over 2 weeks, which can be explained by the formation of the basement membrane components or the apical mucous layer. Caco-2 cells express components of laminin-111 and laminin-511. A synchronous increase in the expression of mucin 3 mRNA (MUC3A/MUC3B) and mucin 17 mRNA (MUC17) and reduced expression of miR-21 and miR-622 microRNA genes were observed. Possible use of the described approach for studying the formation of extracellular matrix is discussed.


Assuntos
Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Membrana Basal/metabolismo , Células CACO-2 , Espectroscopia Dielétrica , Impedância Elétrica , Humanos , MicroRNAs/genética , Mucina-3/genética
6.
Mol Biol (Mosk) ; 52(4): 667-674, 2018.
Artigo em Russo | MEDLINE | ID: mdl-30113032

RESUMO

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.


Assuntos
Diferenciação Celular/genética , Neoplasias Colorretais/genética , Transportadores de Sódio Acoplados à Vitamina C/genética , Transportador 8 de Zinco/genética , Células CACO-2 , Técnicas de Cultura de Células , Membrana Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Impedância Elétrica , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos
7.
Bull Exp Biol Med ; 164(6): 749-752, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29666964

RESUMO

We studied expression profile of microRNA and their target genes in human umbilical vein endothelial cells (HUVEC) during proinflammatory activation with TNFα. TNFα-induced activation of HUVEC was accompanied by a decrease in the expression of CDKN1B, HIST1H3D, and OIP5 genes that are the common target genes for mature microRNA encoded by MIR221, MIR222, and MIR181B1 genes, whose expression increases in activated cells. Proteins encoded by HIST1H3D and OIP5 genes are associated with chromatin compaction and cell cycle. Our results suggest that fetal endothelial microRNA can appear in the maternal blood during various pathological states, e.g., under conditions of preeclampsia.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , MicroRNAs/genética , Fator de Necrose Tumoral alfa/farmacologia , Sítios de Ligação , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação , MicroRNAs/agonistas , MicroRNAs/metabolismo , Modelos Biológicos , Cultura Primária de Células , Transdução de Sinais
8.
Bull Exp Biol Med ; 164(6): 757-761, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29658079

RESUMO

Endothelial HUVEC cells used as an in vitro model of the endothelial monolayer in placental barrier were activated by TNFα in a dose of 2 ng/ml for 24 h. Significant changes in the expression of genes of the SLC family transport protein were observed: an increase in the expression of SLC7A2, SLC12A2, SLC9B2, SLC25A37, SLC16A9, and SLC41A2 and a decrease in the expression of SLC40A1. These transporters participate in the transport of iron, magnesium, sodium, potassium, and chloride ions, protons, and amino acids. It was also found that SLC7A2, SLC12A2, SLC9B2, SLC25A37, and SLC41A2 genes have binding sites for transcriptional factor RelB that together with NFKB2 is the main effector of the non-canonical NF-κB pathway. The expression of RELB and NFKB2 genes was also significantly enhanced in TNFα-activated HUVEC cells, which can attest to the important role of the non-canonical NF-κB pathway in the regulation of gene expression of transport proteins in response to TNFα stimulation.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Subunidade p52 de NF-kappa B/genética , Fator de Transcrição RelB/genética , Fator de Necrose Tumoral alfa/farmacologia , Sistemas de Transporte de Aminoácidos Básicos/agonistas , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Transporte Biológico/efeitos dos fármacos , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade p52 de NF-kappa B/agonistas , Subunidade p52 de NF-kappa B/metabolismo , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Fator de Transcrição RelB/agonistas , Fator de Transcrição RelB/metabolismo
9.
Bull Exp Biol Med ; 164(5): 650-654, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29577195

RESUMO

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.


Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias da Mama/genética , Caderinas/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Modelos Biológicos
10.
Bull Exp Biol Med ; 164(5): 688-692, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29582205

RESUMO

IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
Biochemistry (Mosc) ; 82(10): 1207-1214, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29037142

RESUMO

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.


Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
Bull Exp Biol Med ; 162(4): 515-519, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28243904

RESUMO

The possibility of interactions between warfarin and dasatinib and their interactions with other drugs metabolized by cytochrome P450 isoform CYP3A4 was demonstrated using a previously created cytochrome P450 substrate-inhibitor panel for preclinical in vitro studies of drug biotransformation on a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). Dasatinib and warfarin are inhibitors of CYP2C19 isoform and hence, can interfere the drugs metabolized by this isoform. Our findings are in line with the data obtained on primary culture of human hepatocytes and suggest that the model can be used in preclinical in vitro studies of drugs.


Assuntos
Anticoagulantes/metabolismo , Antineoplásicos/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Dasatinibe/metabolismo , Inativação Metabólica/efeitos dos fármacos , Modelos Biológicos , Varfarina/metabolismo , Anticoagulantes/farmacologia , Antineoplásicos/farmacologia , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Dasatinibe/farmacologia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Dispositivos Lab-On-A-Chip , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Especificidade por Substrato , Varfarina/farmacologia
14.
Biochimie ; 133: 74-79, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28007502

RESUMO

HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 µM concentrations for all studied derivatives of branched tail oxyquinolines.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia/biossíntese , Oxiquinolina/química , Acetamidas/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxiquinolina/farmacologia , Relação Estrutura-Atividade
15.
Bull Exp Biol Med ; 162(1): 170-174, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27882460

RESUMO

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.


Assuntos
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Dispositivos Lab-On-A-Chip , Bupropiona/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2B6/análise , Citocromo P-450 CYP2C19/análise , Citocromo P-450 CYP2C9/análise , Citocromo P-450 CYP3A/análise , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Cetoconazol/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Espectrometria de Massas , Mefenitoína/análogos & derivados , Mefenitoína/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Omeprazol/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/farmacologia , Especificidade por Substrato , Sulfafenazol/farmacologia , Testosterona/metabolismo , Tolbutamida/metabolismo
16.
Bull Exp Biol Med ; 161(3): 425-9, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27496037

RESUMO

We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.


Assuntos
Fígado/citologia , Reatores Biológicos , Sobrevivência Celular/fisiologia , Hepatócitos/citologia , Humanos , Microcirculação/fisiologia
17.
Protein Expr Purif ; 113: 85-93, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25986322

RESUMO

Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 µg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 µM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).


Assuntos
Escherichia coli/genética , Peroxidases/química , Peroxidases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Hemina , Concentração de Íons de Hidrogênio , Corpos de Inclusão , Peroxidases/genética , Peroxidases/isolamento & purificação , Redobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ureia
18.
Biochimie ; 115: 71-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25957835

RESUMO

Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis.


Assuntos
Aminoácidos Aromáticos , Mutagênese Sítio-Dirigida , Peroxidases/química , Peroxidases/metabolismo , Benzotiazóis/metabolismo , Biocatálise , Estabilidade Enzimática , Peróxido de Hidrogênio/metabolismo , Cinética , Modelos Moleculares , Oxirredução , Peroxidases/genética , Conformação Proteica , Redobramento de Proteína , Especificidade por Substrato , Ácidos Sulfônicos/metabolismo , Temperatura
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