RESUMEN
Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl-LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl-LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl-LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl-LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl-LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. Insitu experiments revealed that an antibody raised against HOCl-LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase-H(2)O(2)-chloride system contributes to apoptosis in T-cells.
Asunto(s)
Apoptosis/fisiología , Ácido Hipocloroso/metabolismo , Lipoproteínas LDL/metabolismo , Linfocitos T/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/farmacología , Células Jurkat , Lipoproteínas LDL/química , Lipoproteínas LDL/farmacología , Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
In lipid metabolism, the liver acts as a buffer for transient energy fluctuations. It temporarily stores fatty acids as triacylglycerol and secretes them as very low density lipoprotein into the circulation when the period of maximum lipid load has passed. The lipolytic enzymes responsible for mobilization of internal lipid stores in the liver have not been identified yet. We introduced active site-directed chemical probes for lipolytic activity profiling in complex mixtures, known as activity-based proteomics, and employed it for global analysis and functional annotation of lipolytic proteins in mouse adipose tissue. Here we report the combined application of two approaches using fluorescent and biotinylated probes for discovery and discrimination of lipolytic and esterolytic enzymes in mouse liver subproteomes. Proteomes labeled with the fluorescent probes were analyzed by 2-DE while proteomes labeled with the biotinylated probe were subjected to avidin-affinity isolation. Of 37 totally identified proteins, 15 were detected using both approaches while 14 and 8 were solely identified by 2-DE and avidin-affinity isolation, respectively. Moreover, 12 enzymes were classified as potential lipases and/or cholesteryl esterases by their reaction with probes specific for the respective activities directly in their proteomes.
Asunto(s)
Esterasas/metabolismo , Lipólisis/fisiología , Hígado/metabolismo , Proteoma/análisis , Animales , Azoles , Biotinilación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Ratones , NitrobencenosRESUMEN
This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Esterasas/síntesis química , Lipasa/síntesis química , Compuestos Organofosforados/síntesis química , Animales , Avidina/química , Fluorescencia , Indicadores y Reactivos , Microscopía Fluorescente , Estructura Molecular , Compuestos Organofosforados/química , Páncreas/química , Esterol Esterasa/química , PorcinosRESUMEN
Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Enzimas/metabolismo , Análisis por Matrices de Proteínas , LipólisisRESUMEN
Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.
Asunto(s)
Esterasas/análisis , Colorantes Fluorescentes/análisis , Lipasa/análisis , Organofosfonatos/análisis , Organofosfonatos/síntesis química , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Esterasas/antagonistas & inhibidores , Esterasas/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Lipasa/antagonistas & inhibidores , Lipasa/metabolismo , Organofosfonatos/metabolismo , Oxadiazoles/químicaRESUMEN
Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.