RESUMEN
S-adenosylhomocysteine (SAH) can induce endothelial dysfunction and activation, contributing to atherogenesis; however, its role in the activation of the inflammatory mediator NFkB has not been explored. Our aim was to determine the role of NFkB in SAH-induced activation of endothelial cells. Furthermore, we examined whether SAH, as a potent inhibitor of S-adenosylmethionine-dependent methyltransferases, suppresses the function of EZH2 methyltransferase to contribute to SAH-induced endothelial cell activation. We found that excess SAH increases the expression of adhesion molecules and cytokines in human coronary artery endothelial cells. Importantly, this up-regulation was suppressed in cells expressing a dominant negative form of the NFkB inhibitor, IkB. Moreover, SAH accumulation triggers the activation of both the canonical and non-canonical NFkB pathways, decreases EZH2, and reduces histone 3 lysine 27 trimethylation. EZH2 knockdown recapitulated the effects of excess SAH on endothelial activation, i.e., it induced NFkB activation and the subsequent up-regulation of adhesion molecules and cytokines. Our findings suggest that suppression of the epigenetic regulator EZH2 by excess SAH may contribute to NFkB activation and the consequent vascular inflammatory response. These studies unveil new targets of SAH regulation, demonstrating that EZH2 suppression and NFkB activation mediated by SAH accumulation may contribute to its adverse effects in the vasculature.
Asunto(s)
Células Endoteliales/inmunología , Proteína Potenciadora del Homólogo Zeste 2/inmunología , Inflamación/inmunología , FN-kappa B/inmunología , S-Adenosilhomocisteína/inmunología , Línea Celular , Humanos , Metilación , Metiltransferasas/inmunología , S-Adenosilmetionina/inmunologíaRESUMEN
A competitive immunoassay for S-adenosyl-L-homocysteine (SAH) has been used in the clinical test for homocysteine via an enzymatic conversion reaction. Since S-adenosyl-l-homocysteine is a relatively unstable compound, we have used peptide library phage display to select a new mimotope peptide that interacts with the anti-SAH antibody. By immobilizing the synthetic peptide on solid phase as a competitive surrogate for SAH, we demonstrate its utility in a competitive ELISA assay. The linear range of the assay for SAH was 0.4-6.4 µM, in good correlation to the conventional assay using an SAH-conjugated plate. Our results show that the mimotope peptide has potential to substitute for SAH in immunoassays.
Asunto(s)
Oligopéptidos/química , S-Adenosilhomocisteína/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Imitación Molecular , Oligopéptidos/inmunología , Biblioteca de Péptidos , Unión Proteica , S-Adenosilhomocisteína/inmunologíaRESUMEN
OBJECTIVE: DNA methylation is known to play an important role in gene transcription and alterations of methylation contribute to the development of certain disorders such as cancer and immunodeficiency. Recent years have found an increasing interest in the role of epigenetic modifications in the etiology of human autoimmune diseases, such as systemic lupus erythromatosus (SLE) and rheumatoid arthritis (RA). DNA methyltransferases (DNMTs) are involved in the epigenetic control of DNA methylation processes. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, have important indicator action of cellular methylation status. The aim of this study is to explore if DNA methylation plays a role in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: DNMT1, DNMT3A, and DNMT3B mRNA expression in peripheral blood mononuclear cells (PBMCs) of adult ITP patients were analyzed by real-time quantitative polymerase chain reaction. Plasma SAM and SAH levels were assayed with reversed-phase high performance liquid chromatography (HPLC). RESULTS: DNMT3A and DNMT3B mRNA expressions were significantly lower in ITP patients than in healthy controls (p < 0.001), while DNMT1 mRNA expression was not significantly different between the two groups (p = 0.774). Plasma SAH concentration was significantly elevated in ITP patients than in healthy controls (p < 0.05), while the plasma SAM and SAM/SAH were not significantly different between the two groups (p = 0.133, p = 0.624 respectively). CONCLUSIONS: Our observations suggest that aberrant DNA methylation status reflected by increased plasma SAH concentration and decreased mRNA expression levels of DNMT3A and 3B are possibly involved in the pathogenesis of ITP although the precise mechanisms need further study.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Leucocitos Mononucleares/enzimología , Púrpura Trombocitopénica Idiopática/genética , S-Adenosilhomocisteína/sangre , Adolescente , Adulto , Anciano , Cromatografía Líquida de Alta Presión , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/sangre , ADN (Citosina-5-)-Metiltransferasas/inmunología , ADN Metiltransferasa 3A , Femenino , Humanos , Masculino , Persona de Mediana Edad , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/inmunología , ARN Mensajero/análisis , ARN Mensajero/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , S-Adenosilhomocisteína/inmunología , S-Adenosilmetionina/sangre , S-Adenosilmetionina/inmunología , ADN Metiltransferasa 3BRESUMEN
To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.
Asunto(s)
Oligonucleótidos/química , ARN/química , S-Adenosilhomocisteína/metabolismo , Adenina/química , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Técnicas Biosensibles , Cationes Bivalentes/farmacología , Cromatografía de Afinidad , Cistationina/química , Biblioteca de Genes , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , ARN/metabolismo , S-Adenosilhomocisteína/inmunología , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/químicaRESUMEN
We evaluated the precision, linearity and accuracy of the Abbott IMx and Bio-Rad (Axis) homocysteine assays. Both assays make use of S-adenosyl-homocysteine hydrolase and excess adenosine, to convert homocysteine to S-adenosylhomocysteine (SAH). A monoclonal anti-SAH antibody is then used to quantify SAH. The IMx assay measures the fluorescence polarization of a conjugated SAH analogue for the final analytical step, whereas the Bio-Rad method uses a microplate enzyme immunoassay (EIA) employing an anti-mouse antibody peroxidase conjugate. The Abbott procedure is completely automated whereas the Bio-Rad EIA is performed manually. Between-run coefficient of variation using commercial controls was 2.6% at 7 micromol/L, 2.5% at 13 micromol/L and 1.7% at 24 micromol/L for the Abbott method, and 19.7% at 6.4 micromol/L, 15.9% at 11.0 micromol/L and 14.5% at 23.4 micromol/L for the Bio-Rad method. Both assays correlated well with a high-performance liquid chromatography (HPLC) procedure for homocysteine: Bio-Rad EIA = 1.03HPLC + 1.0 micromol/L, r=0.98, s(y/x)=0.51; Abbott IMx = 1.02HPLC + 0.7 micromol/L, r=0.99, s(y/x) = 0.33. Both methods were linear up to 50 micromol/L homocysteine. The IMx assay had superior precision as well as the technological advantage of being completely automated. Both immunoassays exhibited greatly improved throughput compared with our existing HPLC method.
Asunto(s)
Homocisteína/sangre , Inmunoensayo/métodos , Cromatografía Líquida de Alta Presión , Polarización de Fluorescencia , Humanos , Juego de Reactivos para Diagnóstico , Análisis de Regresión , Reproducibilidad de los Resultados , S-Adenosilhomocisteína/inmunología , S-Adenosilhomocisteína/metabolismo , Sensibilidad y EspecificidadRESUMEN
APO-1/Fas (CD95) is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types. Here we show that L929 cells, expressing human APO-1 treated with agonistic antibodies (anti-APO-1), elicit an early and transient increase of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions. In contrast, anti-APO-1 did not induce an AdoHcy increase in L929-APO-1 Delta4 cells expressing a C-terminally truncated APO-1 lacking part of the 'death domain' known to be required for the transduction of apoptotic signals. Addition of adenosine and D, L-homocysteine also led to an increase of cellular AdoHcy thus enhancing anti-APO-1-induced killing of L929-APO-1 cells. Treatment with anti-APO-1 also induced release of arachidonic acid from phospholipids: this effect was augmented by elevated levels of AdoHcy. In contrast, AdoHcy had only a minor effect on anti-APO-1-mediated DNA fragmentation. These findings suggest that AdoHcy functions as a physiological modulator of APO-1-mediated cell death in L929 cells and enhances anti-APO-1-induced cell killing at least partially by acting via the phospholipase A2 pathway.