Estudio de modificaciones postraduccionales de histonas y su efecto citotóxico sobre células endoteliales

[ES] En los últimos años se ha descrito la participación de las histonas en procesos extracelulares implicados en la inflamación. Las histonas, liberadas de forma controlada por neutrófilos, participan en la inmovilización y eliminación de patógenos por las células del sistema inmunológico. Además, las propias histonas presentan un efecto citotóxico mediado por mecanismos aún no esclarecidos que facilitan el proceso inflamatorio y la respuesta inmunológica. Además, la presencia de histonas produce efectos nocivos sobre las células del endotelio de los vasos sanguíneos, por lo que las histonas circulantes se han visto implicadas, por ejemplo, en procesos de sepsis. En enfermedades con carácter autoinmune como el lupus sistémico eritomatroso (LSE) se han encontrado autoanticuerpos circulantes con especificidad hacia histonas, nucleosomas y otras proteínas nucleares; sin embargo, hasta la fecha se desconoce qué tipo de modificaciones postraduccionales concretas sufren las histonas implicadas en enfermedad, más allá de una participación de histonas citrulinadas o hiperacetiladas en LSE. En el presente trabajo se analizarán los patrones de modificaciones presentes en histonas obtenidas a partir de tejidos de ratones modelo de LSE, y se testeará el efecto citotóxico específico de histonas sometidas a modificaciones similares sobre células endoteliales humanas.[EN] Participation of histones in extracellular processes related to inflammation has been described in recent years. Through their release in a controlled manner by neutrophils, histones take part in the immobilization and elimination of pathogens by immune cells, being key components in the so-called neutrophil extracellular traps (NETs). Besides, histones themselves show a cytotoxic effect induced by mechanisms not fully understood, that contributes to the inflammatory processes and immune responses. However, the presence of histones yields at the same time noxious effects on the endothelial cells of blood vessels, in such a way that plasma histones have been demonstrated to be involved in pathological processes like sepsis, in which prolonged inflammatory response occurs. Nevertheless, which kind of specific postranslational modifications in histones are implicated in disease remains currently unknown, with the exception of the presence of some citrullinated or hyperacetylated histones in systemic lupus erythematosus, for instance. Postranslational modifications of histones contribute to the modulation of gene expression either by allowing or blocking the access to chromatin for the transcription machinery, being one of the key mechanisms underlying epigenetic regulation and, hence, of great relevance for the study of cellular responses to specific environmental conditions. In the present work, the modification pattern present in histones obtained from human cell extracts will be analyzed, and a purification methodology for histones with specific postranslational modifications will be set, in order to evaluate the differences in the cytotoxic capability of the distinct histone types, and the diverse postranslational modifications they might show, on human endothelial cells.Voltolini González, D. (2016). Estudio de modificaciones postraduccionales de histonas y su efecto citotóxico sobre células endoteliales. http://hdl.handle.net/10251/71886.TFG

Vanishing white matter: deregulated integrated stress response as therapy target

Objective: Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation during the integrated stress response (ISR). Mutations reduce eIF2B activity. VWM pathomechanisms remain unclear. In contrast with the housekeeping function of eIF2B, astrocytes are selectively affected in VWM. One study objective was to test our hypothesis that in the brain translation of specific mRNAs is altered by eIF2B mutations, impacting primarily astrocytes. The second objective was to investigate whether modulation of eIF2B activity could ameliorate this altered translation and improve the disease. Methods: Mice with biallelic missense mutations in eIF2B that recapitulate human VWM were used to screen for mRNAs with altered translation in brain using polysomal profiling. Findings were verified in brain tissue from VWM patients using qPCR and immunohistochemistry. The compound ISRIB (for “ISR inhibitor”) was administered to VWM mice to increase eIF2B activity. Its effect on translation, neuropathology, and clinical signs was assessed. Results: In brains of VWM compared to wild-type mice we observed the most prominent changes in translation concerning ISR mRNAs; their expression levels correlated with disease severity. We substantiated these findings in VWM patients’ brains. ISRIB normalized expression of mRNA markers, ameliorated brain white matter pathology and improved motor skills in VWM mice. Interpretation: The present findings show that ISR deregulation is central in VWM pathomechanisms and a viable target for therapy