Identificación de dianas nucleares de nitración durante el estrés por cadmio en Arabidopsis
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2021-02-24
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[ES]El óxido nítrico (NO) es una molécula señal implicada en la respuesta de la planta frente a diferentes condiciones de
estrés biótico y abiótico. El NO y otras especies de nitrógeno reactivo pueden mediar en los procesos de señalización a
través de las modificaciones post-traduccionales, tales como la S-nitrosilación y la nitración de proteínas. Estás
modificaciones post-traduccionales alteran un amplio rango de parámetros funcionales como la actividad catalítica de
la proteína, la localización sub-celular y la estabilidad de las proteínas. Dada la escasez de información respecto a la
nitración de proteínas nucleares en plantas, el objetivo principal de este trabajo consiste en identificar las dianas
nucleares de nitración en cultivos celulares de Arabidopsis thaliana tratados con cadmio. Para ello, se determinarán los
niveles de los marcadores de estrés oxidativo como el peróxido de hidrógeno y malondialdehído mediante
espectrofotometría en cultivos celulares tratados con cadmio, la actividad de las principales enzimas antioxidantes, se
identificarán las proteínas nucleares por espectrometría de masas, se realizará la inmunoprecipitación de proteínas
nucleares empleando un anticuerpo frente a 3-nitrotirosina conjugado con perlas magnéticas y se identificarán las
dianas nitradas por espectrometría de masas. Por último, se realizará un análisis de la modulación funcional de las
dianas identificadas.
[EN]Nitric oxide (NO) is a signal molecule involved in the plant's response to different biotic and abiotic stress conditions. NO and other reactive nitrogen species can mediate signaling processes through post-translational modifications, such as S-nitrosylation and nitration of proteins. These post-translational modifications alter a wide range of functional parameters such as the catalytic activity of the protein, the sub-cellular localization and the stability of the proteins. Given the scarcity of information regarding the nitration of nuclear proteins in plants, the main objective of this work is to identify the nuclear nitration targets in cell cultures of Arabidopsis thaliana treated with cadmium. To do this, the levels of oxidative stress markers such as hydrogen peroxide and malondialdehyde will be determined by spectrophotometry in cell cultures treated with cadmium, the activity of the main antioxidant enzymes, nuclear proteins will be identified by mass spectrometry, the Immunoprecipitation of nuclear proteins using a 3-nitrotyrosine antibody conjugated to magnetic beads and nitrated targets will be identified by mass spectrometry. Finally, an analysis of the functional modulation of the identified targets will be carried out.
[EN]Nitric oxide (NO) is a signal molecule involved in the plant's response to different biotic and abiotic stress conditions. NO and other reactive nitrogen species can mediate signaling processes through post-translational modifications, such as S-nitrosylation and nitration of proteins. These post-translational modifications alter a wide range of functional parameters such as the catalytic activity of the protein, the sub-cellular localization and the stability of the proteins. Given the scarcity of information regarding the nitration of nuclear proteins in plants, the main objective of this work is to identify the nuclear nitration targets in cell cultures of Arabidopsis thaliana treated with cadmium. To do this, the levels of oxidative stress markers such as hydrogen peroxide and malondialdehyde will be determined by spectrophotometry in cell cultures treated with cadmium, the activity of the main antioxidant enzymes, nuclear proteins will be identified by mass spectrometry, the Immunoprecipitation of nuclear proteins using a 3-nitrotyrosine antibody conjugated to magnetic beads and nitrated targets will be identified by mass spectrometry. Finally, an analysis of the functional modulation of the identified targets will be carried out.