RESUMO
5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.
Assuntos
Adenosina/análogos & derivados , Proteínas Arqueais/química , Cisteína/química , Dissulfetos/química , Purina-Núcleosídeo Fosforilase/química , Sulfolobus solfataricus/química , Tionucleosídeos/química , Adenosina/química , Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sulfolobus solfataricus/enzimologia , Termodinâmica , Tionucleosídeos/metabolismoRESUMO
The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Escherichia coli/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Sulfolobus solfataricus/enzimologia , Vidarabina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Arabinonucleosídeos/química , Arabinonucleosídeos/metabolismo , Proteínas Arqueais/química , Ligação Competitiva , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Domínios Proteicos , Purina-Núcleosídeo Fosforilase/química , Especificidade por Substrato , Vidarabina/química , Vidarabina/metabolismoRESUMO
Oxidative stress plays a major role in ethanol-induced liver damage, and agents with antioxidant properties are promising as therapeutic opportunities in alcoholic liver disease. In the present work, we investigated the effect of S-adenosylmethionine (AdoMet), Tyrosol (Tyr), and their combination on HepG2 cells exposed to ethanol exploring the potential molecular mechanisms. We exposed HepG2 cells to 1 M ethanol for 4 and 48 h; thereafter, we recorded a decreased cell viability, increase of intracellular reactive oxygen species (ROS) and lipid accumulation, and the release into culture medium of markers of liver disease such as triacylglycerol, cholesterol, transaminases, albumin, ferritin, and homocysteine. On the other hand, AdoMet and Tyrosol were able to attenuate or antagonize these adverse changes induced by acute exposure to ethanol. The protective effects were paralleled by increased Sirtuin 1 protein expression and nuclear translocation and increased ERK1/2 phosphorylation that were both responsible for the protection of cells from apoptosis. Moreover, AdoMet increased p53 and p21 expression, while Tyrosol reduced p21 expression and enhanced the expression of uncleaved caspase 3 and 9, suggesting that its protective effect may be related to the inhibition of the apoptotic machinery. Altogether, our data show that AdoMet and Tyrosol exert beneficial effects in ethanol-induced oxidative stress in HepG2 cells and provide a rationale for their potential use in combination in the prevention of ethanol-induced liver damage.
Assuntos
Etanol/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Substâncias Protetoras/farmacologia , S-Adenosilmetionina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Células Hep G2 , Humanos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Álcool Feniletílico/farmacologia , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Among nutraceuticals, polyphenols represent the most intriguing and studied class of compounds that can be therapeutics for a large spectrum of the most common diseases, including cancer. Although polyphenols are well known as potent antioxidants, a pro-oxidant effect has been associated with a pro-apoptotic function of these compounds in various types of tumor cells. Annurca apple, a southern Italian variety, is characterized by an extremely high content of polyphenols and displays a stronger antioxidant activity compared with other varieties. In the present study we explored the antiproliferative effect of Annurca apple polyphenol extract (APE) in human breast cancer MCF-7 cells and we investigated the underlying mechanisms. Results showed that at 500 µM catechin equivalent (EqC) APE acts as a pro-oxidant increasing thiobarbituric acid-reactive species cell content of approximately 6-fold more than the untreated cells. We found that APE strongly inhibits the proliferation of MCF-7 cells by inducing G2/M cell cycle arrest and apoptosis. Immunoblot analysis demonstrated that APE treatment increases the levels of p53 and p21, downregulates the expression of the cell cycle regulatory protein cyclin D1, and inhibits ERK1/2 phosphorylation. Moreover, APE treatment caused a marked increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by caspase-9, -6, -7, and poly(ADP ribose) polymerase cleavage. Altogether our data indicate that APE, at elevated concentrations, acts as a potent pro-oxidant and antiproliferative agent able to downregulate ERK1/2 pathway leading to cell cycle inhibition and apoptosis and provides a rationale for its potential use in the development of novel therapeutics towards breast cancer.