María A. Pajares

S-adenosylmethionine synthesis: Molecular mechanisms and clinical implications

Methionine adenosyltransferase (MAT) is an ubiquitous enzyme that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. In mammals, there are two genes coding for MAT, one expressed exclusively in the liver and a second enzyme present in all tissues. Molecular studies indicate that liver MAT exists in two forms: as a homodimer and as a homotetramer of the same oligomeric subunit. The liver-specific isoenzymes are inhibited in human liver cirrhosis, and this is the cause of the abnormal metabolism of methionine in these subjects.

Effects of S-adenosylmethionine on lipid peroxidation and liver fibrogenesis in carbon tetrachloride-induced cirrhosis.

BACKGROUND/AIM The aim of this study was to investigate the effects of S-adenosylmethionine on liver peroxidation and liver fibrogenesis in carbon tetrachloride-induced cirrhosis. METHODS Cirrhosis was induced in three groups of six rats by repeated injections of carbon tetrachloride over 9 weeks. One group of animals was treated only with carbon tetrachloride, and the other two received carbon tetrachloride plus S-adenosylmethionine (10 mg/kg intramuscularly daily) from week 3 to week 9, and from week 6 to week 9 of the study, respectively. Two additional groups of six rats, a control group and a group treated only with S-adenosylmethionine, were also studied. Glutathione concentration, thiobarbituric acid-reactive substances, collagen content, prolyl hydroxylase activity, and procollagen type I mRNA expression were determined in liver samples. RESULTS All carbon tetrachloride-treated rats had cirrhosis at the end of the study. Cirrhosis was also present in five of the six carbon tetrachloride-treated rats receiving S-adenosylmethionine for 3 weeks, but in only one of the six rats that received S-adenosylmethionine for 6 weeks. Hepatic glutathione was significantly diminished in carbon tetrachloride-treated rats (2.7 +/- 0.3 mumol/g tissue) and returned to normal in rats receiving S-adenosylmethionine for 3 or 6 weeks (3.7 +/- 0.13 and 3.9 +/- 0.11 mumol/g tissue, respectively). The hepatic thiobarbituric acid-reactive substances were significantly lower in rats treated with carbon tetrachloride and S-adenosylmethionine for 6 weeks (98 +/- 5 nmol/g) than in rats treated with carbon tetrachloride (134 +/- 12 nmol/g) and in those treated with carbon tetrachloride and S-adenosylmethionine for 3 weeks (127 +/- 13 nmol/g). There were no differences in either hepatic collagen and prolyl hydroxylase activity between rats that received only carbon tetrachloride and those treated with S-adenosylmethionine for 3 weeks. In contrast, carbon tetrachloride-treated rats receiving S-adenosylmethionine for 6 weeks had significantly lower collagen and prolyl hydroxylase activity than the other two groups. A much greater increase in procollagen type I mRNA was found in carbon tetrachloride-treated rats than in rats treated with carbon tetrachloride and S-adenosylmethionine for 6 weeks. Furthermore, there was a significant correlation between the hepatic thiobarbituric acid-reactive substances and prolyl hydroxylase activity and hepatic collagen. CONCLUSIONS We conclude that the early administration of S-adenosylmethionine in a model of carbon tetrachloride-induced liver injury restores glutathione levels and reduces lipid peroxidation, resulting in less advanced liver fibrosis.

Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione

Resumen del poster presentado al 50th Inner Ear Biology Workshop, celebrado en Alcala de Henares-Madrid (Espana) del 10 al 13 de septiembre de 2013.Resumen del trabajo presentado al 15o Congreso Nacional de la Sociedad Espanola de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013.Resumen del poster presentado al CIBERDEM Annual Meeting, celebrado en Cerdanyola del Valles, Barcelona (Espana) del 11 al 13 de mayo de 2016.-- et al.Resumen del trabajo presentado al XXXVIII Congreso de la Sociedad Espanola de Ciencias Fisiologicas (SECF), celebrado en Zaragoza del 13 al 16 de septiembre de 2016.Poster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los dias 3 al 6 de julio de 2013 en Berlin (Alemania)Memoria presentada para optar al grado de Doctor por la Licenciada en Biologia Angela Prieto Folgado y realizada en el Instituto de Investigaciones Biomedicas Alberto Sols.La realizacion de este trabajo ha sido posible gracias a la financiacion otorgada por el FIS al proyecto de investigacion 96/1803.Grant Funding Source: Supported by the Fondo de Investigaciones Sanitarias (PI0011406) to MF.The chemotherapeutic study of a limited series of steroidal sapogenins from several endemic species of the flora of the Canary Islands is presented here. On the whole, they possess a very weak antibacterial activity, a slight antifungal effect and one of them, vespertilin, displays interesting cytostatic activity (ID50 = 5 micrograms/ml). A pharmacodynamic screening carried out on this product mainly revealed very slight toxicity, antihistaminic activity and a light tranquilizing effect. The data obtained justify further research.The purpose of this study was to characterize the role of ions other than Ca2+ in hepatic responses to alpha 1-adrenergic stimulation. We report that the alpha 1-adrenoreceptor activation of hepatic functions is accompanied by extracellular acidification and an increase in intracellular pH. These effects are dependent on extracellular Na+ concentration and are inhibited by the Na+/H+ antiporter blocker 5-(N-ethyl-N-isopropyl) amiloride under conditions that preclude antagonistic effects on agonist binding. Thus, the activation of plasma membrane Na+/H+ exchange is an essential feature of the hepatic alpha-adrenoreceptor-coupled signaling pathway. The following observations indicate that the sustained hepatic alpha 1-adrenergic actions rely on a functional coupling between the plasma membrane Na+/H+ and Na+/Ca2+ exchangers, resulting in the stimulation of Ca2+ influx. 1) Inhibition of the Na+/K(+)-ATPase does not prevent the alpha 1-adrenergic effects. However, alpha 1-adrenoreceptor stimulation fails to induce intracellular alkalinization and to acidify the extracellular medium in the absence of extracellular Ca2+. 2) A non-receptor-induced increase in intracellular Na+ concentration, caused by the ionophore monensin, stimulates Ca2+ influx and increases vascular resistance. 3) Inhibition of Na+/Ca2+ exchange prevents, in a concentration-dependent manner, most of the alpha 1-agonist-induced responses. 4) The actions of Ca(2+)-mobilizing vasoactive peptide receptors or alpha 2-adrenoreceptors, which produce neither sustained extracellular acidification nor release of Ca2+, are insensitive to Na+/H+ exchange blockers.Poster presentado en la VII Reunion Anual de la Red Tematica de Investigacion Cooperativa en Cancer (RTICC), celebrada en Salamanca el 24 de septiembre de 2014Resumen del trabajo presentado al VI Meeting de la Red Espanola de Canales Ioniocs (RECI), celebrado en Santiago de Compostela del 6 al 8 de septiembre de 2017.Tesis Doctoral presentada por Laura Jimenez Perez para optar al grado de doctor por la Universidad de Valladolid, Departamento de Bioquimica y Biologia Molecular y FisiologiaPoster presentado en la VII Reunion Anual de la Red Tematica de Investigacion Cooperativa en Cancer (RTICC), celebrada en Salamanca el 24 de septiembre de 2014Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.Esta Tesis Doctoral fue realizada en el Centro Andaluz de Biologia del Desarrollo por la licenciada Briseida Beli Cacho Valadez para optar al grado de Doctor por la Universidad Pablo de Olavide.Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.Trabajo presentado en el XI Simposi de Neurobiologia: Future technical advances, organizado por la Socitat Catalana de Biologia, en Barcelona, los dias 12 y 13 de noviembre de 2018El estudio de la relacion entre componentes de la dieta y la salud/enfermedad utiliza metodos de valoracion de la ingesta dietetica, del estatus nutricional y de marcadores de funcion o de efecto. En concreto, en el estudio de los carotenoides y la salud ocular, interesa el estudio de dos carotenoides sin actividad provitamina A, la luteina y la zeaxantina, por su posible papel en la optimizacion de la funcion visual y en la prevencion de enfermedades cronicas asociadas a la edad, y de tres carotenoides con actividad provitamina A: -caroteno, -caroteno y -criptoxantina, por ser precursores de retinol, nutriente del que depende el ciclo visual para una vision normal. En el presente trabajo se ha llevado a cabo el estudio de los carotenoides de la dieta mas relevantes para la salud ocular humana considerando de forma simultanea parametros relacionados con la ingesta, el estatus y la funcion visual, asi como diversas variables que pueden modificar el estatus nutricional, como son la concentracion de lipidos en sangre, y la bioaccesibilidad de los carotenoides a partir de alimentos de amplio consumo...Fetal rat hepatocytes treated with transforming growth factor beta (TGF-beta) die by apoptosis. However, a subpopulation of them survives and undergoes an epithelial mesenchymal transition (EMT). This transition also occurs upon incubation with fetal bovine serum. We have isolated the subpopulations that undergo EMT (TGF-beta-treated-fetal hepatocytes: TbetaT-FH; serum-treated-fetal hepatocytes: ST-FH) and show that they present high levels of vimentin and Snail expression and lack cytokeratin 18 and E-cadherin. Both TbetaT-FH and ST-FH cells require mitogens to grow and maintain the response to TGF-beta in terms of growth inhibition. However, they lack differentiation markers such as the liver-enriched transcription factors hepatocyte nuclear factor 4 (HNF-4) or HNF-1alpha and express the progenitor marker OV-6. Interestingly, the EMT process confers them resistance to the apoptotic effect of TGF-beta, with cells showing higher levels of active AKT and Bcl-x(L) than fetal hepatocytes. In summary, these cells are refractory to the apoptotic effects of TGF-beta, showing characteristics of liver progenitors and of some hepatocellular carcinoma cells.Memoria de tesis presentada por Luis Vazquez Fonseca, Licenciado en Bioquimica para optar al grado de Doctor. Esta Tesis Doctoral ha sido realizada bajo el programa de doctorado de Biotecnologia y Tecnologia Quimica en el grupo de investigacion del CIBERER U729 en el Centro Andaluz de Biologia del Desarrollo, Area de Biologia Celular del Departamento de Fisiologia, Anatomia y Biologia Celular de la Universidad Pablo de Olavide y bajo la direccion del Dr. Carlos Santos Ocana y el Dr. Placido NavasResumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Poster presentado al 17o Congreso Nacional de la Sociedad Espanola de Neurociencia, celebrado en Alicante del 27 al 30 de septiembre de 2017.The mutations at the bithorax locus produce a transformation of anterior haltere into anterior wing. The bx1 allele presents unusual features when compared with other bx alleles. The phenotype of bx1 homozygotes is temperature sensitive but only with regard to the distal and not to the proximal transformation, thus suggesting two different components in the bithorax transformation. The phenotype of bx1 homozygotes is stronger than that of bx1 over the deletion of the gene, suggesting a trans interaction of the bx1 chromosomes which results in mutual partial inactivation. We show by temperature shift and clonal analysis experiments that the decision on whether to differentiate haltere or wing structures is taken at the end of the proliferation period of the mutant disc.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Poster presentado al XXVII Congreso Nacional de la Asociacion Espanola de Genetica Humana celebrado en Madrid del 10 al 12 de abril de 2013.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Poster presentado en el XI European Meeting on Glial Cells in Health and Disease, celebrado los dias 3 al 6 de julio de 2013 en Berlin (Alemania)Resumen del trabajo presentado al Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio de 2019.Poster presentado en el XII European Meeting on Glial Cells in Health and Disease, celebrado los dias 15 a 18 de julio de 2015 en Bilbao (Espana)Trabajo presentado en el XL Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular. FEBS3+1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular, celebrado en Barcelona (Espana), del 23 al 26 de octubre de 2017Resumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Trabajo presentado en el XII GEIRLI Meeting: New trends in redox biology: a multidisciplinary approach, celebrado en Barcelona (Espana), los dias 4 y 5 de julio de 2019Treatment of nucleosomal particles with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by a biphasic release of histones H2A plus H2B; one H2A.H2B dimer is more easily released than the other. This behavior allows the preparation of nucleosomal particles containing only one H2A.H2B dimer, which were complemented with 125I-labeled H2A.H2B. These reconstituted particles, which contain one labeled and one unlabeled H2A.H2B dimer, were treated with the amount of reagent needed to release one of the two H2A.H2B dimers. Radioactivity was equally distributed between residual particles and released proteins, which is consistent with equivalent binding sites in the nucleosomal particle for H2A.H2B dimers, rather than with intrinsically different sites. The asymmetric release of H2A.H2B dimers would be caused by a change in the binding site of one dimer following the release of the other. This behavior might be related to the structural dynamics of nucleosomes.Resumen del trabajo presentado al European Society of Cardiology (ESC) Congress, celebrado en Barcelona (Espana) del 26 al 30 de agosto de 2017.Resumen del poster presentado al 49th European Association for the Study of Diabetes Annual Meeting, celebrado en Barcelona (Espana) del 23 al 27 de septiembre de 2013.-- et al.Trabajo doctoral realizado por Da Rebeca Lapresa Ruiz de Gauna, para optar al grado de doctor por la Universidad de Salamanca.Rationale: Several animal models have been developed to study acute lung injury (ALI); however the majority of these studies are focused on different mechanisms within the acute phase. These models do not allow studying the mechanisms in the later phases or testing any possible long-term treatment. The aim of this study was to develop an experimental ALI model simulating bronchial aspiration of gastric contents with bacterial superinfection with alveolar epithelial damage persisting over time. Methods: Sprague-Dawley rats (200-250g) were anesthetized with isofluorane. ALI was induced by intratracheal instillation of HCl (1 µl/g, 0.1 mol/L pH=1.4) followed by instillation of LPS from Escherichia coli O55:B5 (0, 10, 20, 30 or 40µg/g b.w.) two hours later. Control rats were treated with intratracheal instillations of saline. After 72h, the animals were sacrificed and bronchoalveolar lavage fluid (BALF) was sampled for further analysis of total protein concentration by bicinchoninic acid method. Results: At 72 h, rats suffered a significant loss of weight proportional to the administered dose of LPS (5.6% with 10µg/g b.w, 12.6% with 20µg/g b.w, 14.2% with 30µg/g b.w and 17.7% with 40µg/g b.w). Control rats gained in weight at 72h. LPS at 10, 20, 30 and 40µg/g b.w induced a 1.7, 2.5, 2.9 and 3.4 fold increase in total protein concentration in BAL fluid, respectively, reflecting a substantial increase proportional to the LPS dose. Conclusion: The degree of weight loss and the increase of total protein concentration in BAL fluid in the current model may reflect disease severity and progression. This model would be useful in future for new therapeutical options. Grant acknowledgements: FIS-PI12/02548 and Fundacio Parc Tauli.Resumen del trabajo presentado al European Respiratory Society (ERS) International Congress, celebrado en Paris (Francia) del 15 al 19 de septiembre de 2018.Resumen del trabajo presentado a las 5as Jornadas de Formacion del CIBERES celebradas en Bunyola (Mallorca) del 18 al 19 de octubre de 2012.Resumen del poster presentado al Joint FEPS & XXXVI Spanish Physiological Society Congress (Sociedad Espanola de Ciencias Fisiologicas) celebrado en Santiago de Compostela (Espana) del 8 al 11 de septiembre de 2012.Resumen del trabajo presentado al XIII Congreso de la Sociedad Espanola del Dolor, celebrado en Pamplona del 2 al 4 de junio de 2016.This work was supported by grants FIS-01/1048 and FIS-02/1199 from the Fondo de Investigacion Sanitaria and grant SA-087/01 from Junta de Castilla y Leon.Resumen del poster presentado al Joint Meeting of the American Physiological Society and the Physiological Society, celebrado en Dublin (Irlanda) del 29 al 31 de julio de 2016.Trabajo presentado al 5th International Conference on Phospholipase A2 Mediated Signaling in Translational Medicine celebrado en New Orleans (US) del 20 al 21 de mayo de 2013.Tesis Doctoral presentada por Rebeca Torres Merino para optar al grado de Doctora por la Universidad de Valladolid, Facultad de Medicina: Dpto. de Bioquimica y Biologia Molecular y Fisiologia.Poster presentado al Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), celebrado en Seattle, Washington (US) del 1 al 5 de mayo de 2016.Resumen del trabajo presentado al 63rd Annual Meeting Biophysical Society, celebrado en Baltimore, Maryland (USA) del 2 al 6 de marzo de 2019.Poster presentado al XXXVII Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Resumen del poster presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.-- Tambien presentado al 15o Congreso Nacional de la Sociedad Espanola de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013.Resumen del trabajo presentado al XXXVI Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular celebrado en Madrid del 4 al 6 de septiembre de 2013.Resumen del trabajo presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.Resumen del poster presentado al XXVIII Congreso Nacional de la Sociedad Espanola de diabetes, celebrado en Bilbao del 20 al 22 de abril de 2016.SAF2016-77703-C2-2-R of the Ministerio de Economia y Competitividad, Spain and the European Regional Development Fund (ERDF); AGAUR 2017-SGR106 and the CERCA Programme of the Generalitat de Catalunya; C. Sanfeliu belong to Group 05 of CIBER Epidemiologia y Salud Publica (CIBERESP) of the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain

Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes stabilized by Cu(II) bridges.

Several lines of evidence have shown glycosaminoglycans (GAGs) to be physiological ligands of the prion protein (PrP), but the molecular and regulatory aspects of the interaction remain unknown. Using full-length recombinant prion protein and low molecular mass heparin and heparan sulfate as glycosaminoglycans, we have found that the interaction occurs with the formation of oligomeric complexes. Within the protein-glycosaminoglycan complexes, PrP exhibited an enhanced fluorescence emission and a reduced solvent exposure. The pH and ionic strength-dependence of the interaction reveals His residues as the main binding sites at acid pH. A synthetic peptide consisting of four octarepeats is able to reproduce the His-dependent binding of the protein, thus demonstrating the role of the octarepeats in the GAG interaction. Alternatively, PrP can bind GAGs through His-bound Cu(II). These Cu(II) bridges promote a tighter interaction, as shown by the increased resistance to ionic strength, to protease action, and to pH-induced cation release. Inspection of other cations shows that Zn(II) but not Ni(II) shares the interaction trend. Taken together, our data suggest that the octarepeat region constitutes a novel GAG-binding sequence and that His-bound Cu(II) may act as a cofactor for intermolecular recognition reactions, allowing the formation of PrP-Cu(II)-glycosaminoglycan assemblies that may be crucial entities in the PrP metabolism.

Structure-function relationships in methionine adenosyltransferases

Abstract.Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.

Conformational signals in the C-terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution

The methyl donor S‐adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues, both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C‐terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel‐filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S‐adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles.—Reytor, E., Pérez‐Miguelsanz, J., Alvarez, L., Pérez‐Sala, D., Pajares, M. A. Conformational signals in the C‐terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution. FASEB J. 23, 3347–3360 (2009). www.fasebj.org

How is rat liver S‐adenosylmethionine synthetase regulated?

The in vivo regulation of S‐adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed.

Cyclopentenone Prostaglandins with Dienone Structure Promote Cross-Linking of the Chemoresistance-Inducing Enzyme Glutathione Transferase P1-1

Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in cancer chemoprevention and chemoresistance and is a key target for anticancer drug development. Oxidative stress or inhibitor-induced GSTP1-1 oligomerization leads to the activation of stress cascades and apoptosis in various tumor cells. Therefore, bivalent glutathione transferase (GST) inhibitors with the potential to interact with GST dimers are been sought as pharmacological and/or therapeutic agents. Here we have characterized GSTP1-1 oligomerization in response to various endogenous and exogenous agents. Ethacrynic acid, a classic GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and diamide all induced reversible GSTP1-1 oligomerization in Jurkat leukemia cells through the formation of disulphide bonds involving Cys47 and/or Cys101, as suggested by reducing and nonreducing SDS-polyacrylamide gel electrophoresis analysis of cysteine to serine mutants. Remarkably, the electrophilic prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induced irreversible GSTP1-1 oligomerization, specifically involving Cys101, a residue present in the human but not in the murine enzyme. 15d-PGJ2-induced GSTP1-1 cross-linking required the prostaglandin (PG) dienone structure and was associated with sustained c-Jun NH2-terminal kinase activation and induction of apoptosis. It is noteworthy that 15d-PGJ2 elicited GSTP1-1 cross-linking in vitro, a process that could be mimicked by other dienone cyclopentenone PG, such as Δ12-PGJ2, and by the bifunctional thiol reagent dibromobimane, suggesting that cyclopentenone PG may be directly involved in oligomer formation. Remarkably, Δ12-PGJ2-induced oligomeric species were clearly observed by electron microscopy showing dimensions compatible with GSTP1-1 tetramers. These results provide the first direct visualization of GSTP1-1 oligomeric species. Moreover, they offer novel strategies for the modulation of GSTP1-1 cellular functions, which could be exploited to overcome its role in cancer chemoresistance.

ROLE OF THIOLTRANSFERASES ON THE MODULATION OF RAT LIVER S-ADENOSYLMETHIONINE SYNTHETASE ACTIVITY BY GLUTATHIONE

Rat liver S‐adenosylmethionine synthetase, high‐ and low‐M r forms, are regulated in vitro by the GSH/GSSG ratio at pH 8. The inhibition and oxidation constants for both forms have been calculated in the presence of thioltransferases. The mechanism of the reaction appeared to involve the formation of intramolecular disulfides. Increases of 3‐ to 4‐fold in the oxidation constants for both S‐adenosylmethionine synthetase isoenzymes in the presence of protein disulfide isomerase suggested the possibility of a thiol‐disulfide exchange regulatory mechanism for this enzyme in vivo. The significance of these results is discussed on the light of the data available relating glutathione changes and modulation of enzyme activities, either in vivo and in vitro.

Characterization of rat liver-specific methionine adenosyltransferase gene promoter. Role of distal upstream cis-acting elements in the regulation of the transcriptional activity.

Methionine adenosyltransferase is a ubiquitous enzyme that catalyzes the only known route of biosynthesis of S-adenosylmethionine, the major methyl group donor in cell metabolism. In mammals, two different methionine adenosyltransferases exist: one is confined to the liver, and the other one is distributed in extrahepatic tissues. In the present study, we report the cloning of the 5′-flanking region of liver-specific methionine adenosyltransferase gene from rat. Two closely spaced sites for transcriptional initiation were identified by primer extension analysis. The major transcription start site was determined to be 29 nucleotides downstream from the putative TATA box. Transient transfection assays of constructs containing sequentially deleted 5′-flanking sequences fused to the luciferase gene showed that rat hepatic methionine adenosyltransferase promoter was able to efficiently drive reporter expression not only in liver-type cells (rat hepatoma H35 cells and human hepatoblastoma HepG2 cells) but also in Chinese hamster ovary cells. Two regions spanning nucleotides −1251 to −958 and −197 to +65 were found to be crucial for the promoter efficiency. The distal upstream region contains elements that positively regulate promoter activity in H35 and HepG2 cells but are ineffective in Chinese hamster ovary cells. Eight protein binding sites were characterized in both regions by DNase I footprinting analysis. Two of these elements, sites A and B, located in the distal region, were found to be essential for the regulation of promoter activity. Electrophoretic mobility shift assays and competition experiments showed that site A is recognized by an NF1 protein. Site B was able to interact with a member of HNF-3 family when nuclear extracts from rat liver and H35 cells were used in the in vitro assay, but an additional binding activity to an NHF1-like protein was obtained with the hepatoma cell extracts. It is suggested that this differential binding can contribute to the cell specificity of promoter function.