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Within the last twenty years the view on reactive oxygen species (ROS) has changed; they are no longer only considered to be harmful but also necessary for cellular communication and homeostasis in different organisms ranging from bacteria to mammals. In the latter, ROS were shown to modulate diverse physiological processes including the regulation of growth factor signaling, the hypoxic response, inflammation and the immune response. During the last 60-100 years the life style, at least in the Western world, has changed enormously. This became obvious with an increase in caloric intake, decreased energy expenditure as well as the appearance of alcoholism and smoking; These changes were shown to contribute to generation of ROS which are, at least in part, associated with the occurrence of several chronic diseases like adiposity, atherosclerosis, type II diabetes, and cancer. In this review we discuss aspects and problems on the role of intracellular ROS formation and nutrition with the link to diseases and their problematic therapeutical issues.

Resumen del trabajo presentado al X Meeting of the Spanish Group for Research on Free Radicals: "Symposium on Oxidative Stress and Redox Signaling in Biology and Medicine", celebrado en la Facultad de Medicina de la Universidad de Valencia (España) del 2 al 4 de junio de 2014.-- et al., The adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by the hypoxia-inducible factors (HIF). There is still a debate on whether the production of reactive oxygen species (ROS) increases in hypoxia, which in turn may contribute to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS are capable of transducing signals by reversibly modifying the redox state of cysteine residues of proteins. We have used different techniques for measuring superoxide production kinetics, and we have observed that acute hypoxia produces a superoxide burst in different types of cells. By using diverse thiol redox proteomics techniques, we have observed an increase in cysteine reversible oxidation in endothelial cells in acute hypoxia. Indeed, we have been able to identify a number of proteins that are specifically oxidised in these conditions. These cysteine oxidation signals may mediate different adaptations to hypoxia, before the HIF pathway is fully activated. We hypothesize that ROS signals can be initiated by increased superoxide production in mitochondria, which can be translated in the oxidation of sensitive cysteine residues in cells subjected to acute hypoxia, mediating further functional adaptations.

Comunicaciones a congresos

Comunicaciones a congresos

Comunicaciones a congresos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 28-11-2018, Esta tesis tiene embargado el acceso al texto completo hasta el 28-05-2020, Las enfermedades de válvula aórtica son las enfermedades valvulares más comunes en los países occidentales, y la principal causa de cirugía de reemplazo valvular. Hay dos presentaciones principales atendiendo a su patogenia: estenosis aórtica degenerativa, que es la más frecuente, e insuficiencia aórtica, que tiene una menor prevalencia. Por otro lado, el ictus isquémico es causado por el taponamiento de un vaso sanguíneo por un coágulo de sangre en el cerebro. Diferenciar la etiología del ictus es fundamental para tratar a los pacientes tras la rehabilitación después del ictus, con el fin de evitar recurrencias. El propósito de este trabajo consiste en estudiar cambios en los niveles de proteínas y modificaciones postraduccionales oxidativas en cisteínas que expliquen las diferencias entre las enfermedades de estenosis e insuficiencia y puedan ofrecer biomarcadores candidatos para diferenciar entre los grupos clínicos. Por otro lado, comparando los proteomas entre trombos de origen cardioembólico y trombos de origen aterotrombótico, las dos etiologías más frecuentes en el ictus isquémico, se podrán encontrar biomarcadores candidatos para discriminar entre ambas etiologías. Con este objetivo se han analizado válvulas aórticas de pacientes con estenosis aórtica, pacientes con insuficiencia aórtica y válvulas de corazones que han sido reemplazados durante un trasplante de órganos en los que se ha verificado que las válvulas funcionan de manera normal. Las muestras de ictus isquémico se obtuvieron de los trombos extraídos mediante trombectomía y clasificadas atendiendo a su etiología: cardioembólicas y aterotrombóticas. Se ha empleado el método Gelsilox con marcaje isobárico para la cuantificación relativa de niveles de proteína así de los péptidos que contienen cisteína en su forma reducida y oxidada. La combinación de la proteómica cuantitativa y la proteómica redox tiólica, así como el uso complementario de diferentes métodos de análisis ha permitido clasificar las muestras de los diferentes grupos clínicos, permitiendo profundizar en los mecanismos patológicos de las enfermedades estudiadas

Memoria presentada por el licenciado en Biología: Pablo Hernansanz Agustín para optar al título de Doctor por la Universidad Autónoma de Madrid. Este trabajo se realizó en el Servicio de Inmunología del Hospital de la Princesa y en el Instituto de Investigaciones Biomédicas “Alberto Sols”, [EN] Oxygen is a key molecule for aerobic life. Eukaryotes have evolved towards an aerobic metabolism which has allowed them to adapt to a plethora of environments and to develop into multicellular systems. The proper distribution of oxygen inside the organism is vital for tissue function and its decreased availability, termed hypoxia, can compromise the survival of the organism. Cells, tissues and organs have developed a series of molecular responses in order to adapt to hypoxia. Mitochondria are the main oxygen consumers in most eukaryotic cells and also a major source of production of reactive oxygen species (ROS) that can be used by the cells to trigger redox cell signaling processes. There has been a long debate on whether cells respond to hypoxia by increasing ROS production and their involvement in different responses to hypoxia. Since superoxide anion is the first ROS formed from oxygen reduction by the mitochondrial electron transport chain (ETC), we have focused on its specific detection in acute hypoxia. We have observed that superoxide anion production by the mitochondria is increased in the first minutes of hypoxia and that it decreases thereafter, reaching basal levels before one hour of hypoxia, what we have called a hypoxic superoxide ‘burst’. In order to dissect the mechanism triggering this response, we have studied the role of different mitochondrial ETC complexes and ion transporters in the production of the hypoxic superoxide burst. We observe that complex I, complex III and the mitochondrial sodium/calcium exchanger (NCLX) are necessary for hypoxic ROS production, and that there could be a chain of events among them. Mitochondrial complex I (CI) undergoes ‘deactivation’ in acute hypoxia; this was inversely proportional to the oxygen concentration and could switch its activity to a sodium/proton antiporter. CI deactivation is necessary for the enhancement of mitochondrial sodium/calcium exchange through NCLX, which in turn promoted depolarization of the mitochondrial inner membrane. Mitochondrial sodium/calcium exchange is necessary for superoxide production in which complex III would be involved, although the exact source of superoxide has not been determined. We have been able to observe the implication of NCLX in a variety of experimental settings, in which NCLX inhibition alter different responses to hypoxia, such as activation of the HIF pathway and hypoxic pulmonary vasoconstriction. We have assessed that this mechanism operates also in neurons and brain tissue and that treatment with a NCLX inhibitor diminishes cell death in a model of stroke, probably by reducing oxidative damage. These results open a new window in mitochondrial redox biology research due to the involvement of complex I deactivation and NCLX in superoxide production, which could also have therapeutic implications., [ES] El oxígeno es una molécula clave para la vida aeróbica. Los organismos eucariotas han evolucionado hacia un metabolismo aeróbico que les ha permitido adaptarse a una plétora de ambientes y desarrollarse como sistemas multicelulares. La adecuada distribución del oxígeno dentro del organismo es vital para la función tisular y la disminución de sus disponibilidad, denominada hipoxia, puede comprometer la supervivencia del organismo. Las células, los tejidos y los órganos han desarrollado una serie de respuestas moleculares que permiten su adaptación a la hipoxia. Las mitocondrias son los principales consumidores de oxígeno en muchas células eucariotas y son también una de las principales fuentes de producción de especies reactivas de oxígeno (ROS) que pueden usar las células para promover procesos de señalización celular redox. Se ha generado un prolongado debate sobre si las células responden a la hipoxia incrementando la producción de ROS y su implicación en las diferentes respuestas a la hipoxia. Dado que el anión superóxido es la primera ROS formada a partir de la reducción del oxígeno por la cadena de transporte electrónico (ETC) mitocondrial, nos hemos centrado en su detección específica en hipoxia aguda. Hemos observado que se incrementa la producción de anión superóxido en las mitocondrias en los primeros minutos de hipoxia, disminuyendo posteriormente hasta llegar a niveles basales antes de una hora de hipoxia, lo que hemos llamado un ‘estallido’ de superóxido en hipoxia. Para diseccionar el mecanismo que desencadena esta respuesta, hemos estudiado el papel de distintos complejos de la ETC y trasportadores mitocondriales de iones. Observamos que el complejo I, el complejo III y el intercambiador sodio/calcio mitocondrial (NCLX) son necesarios para la producción de ROS en hipoxia, y que puede haber una cadena de eventos entre ellos. El complejo I (CI) mitocondrial se desactiva en hipoxia aguda, de manera inversamente proporcional a la concentración de oxígeno, y puede cambiar su actividad a la de antiportador sodio/protón. La desactivación de CI es necesaria para activar el intercambio sodio/calcio a través de NCLX, lo que a su vez promueve la despolarización de la membrana mitocondrial interna. El intercambio sodio/calcio en la mitocondria es necesario para la producción de superóxido, en lo que está implicado el complejo III, si bien al fuente exacta de superóxido no se ha determinado. Hemos podido observar la implicación de NCLX en una variedad de escenarios experimentales, en los que la inhibición de NCLX altera diferentes respuestas a la hipoxia, como la activación de la ruta de HIF y la vasoconstricción pulmonar hipóxica. Hemos comprobado que este mecanismo funciona también en neuronas y tejido cerebral, y que el tratamiento con un inhibidor de NCLX reduce la muerte celular en un modelo de ictus, probablemente disminuyendo el daño oxidativo. Estos resultados abren una nueva ventana en la biología redox mitocondrial debido a la implicación de la desactivación del complejo I y de NCLX en la producción de superóxido, l que puede tener también implicaciones terapéuticas

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1,2,3,4, a phenomenon that occurs in hypoxia4,5,6,7,8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism., This research has been financed by Spanish Government grants (ISCIII and AEI agencies, partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R. and J.A.E.); CP07/00143, PS09/00101, PI12/00875, PI15/00107 and RTI2018-094203-B-I00 (to A.M.-R.); CP12/03304 and PI15/01100 (to L.M.); CP14/00008, CPII19/00005 and PI16/00735 (to J.E.); SAF2016-77222-R (to A. Cogolludo); PI17/01286 (to P.N.); SAF2015-65633-R, RTI2018-099357-B-I00 and CB16/10/00282 (to J.A.E.); RTI2018-095793-B-I00 (to M.G.L.); and SAF2017-84494-2-R (to J.R.-C.), by the European Union (ITN GA317433 to J.A.E. and MC-CIG GA304217 to R.A.-P.), by grants from the Comunidad de Madrid B2017/BMD-3727 (to A. Cogolludo) and B2017/BMD-3827 (to M.G.L.), by a grant from the Fundación Domingo Martínez (to M.G.L. and A.M.-R.), by the Human Frontier Science Program grant HFSP-RGP0016/2018 (to J.A.E.), by grants from the Fundación BBVA (to R.A.-P. and J.R.-C.), by the UCM-Banco Santander grant PR75/18-21561 (to A.M.-d.-P.), by the Programa Red Guipuzcoana de Ciencia, Tecnología e Información 2018-CIEN-000058-01 (to J.R.-C.) and from the Basque Government under the ELKARTEK Program (grant no. KK-2019/bmG19 to J.R.-C.), by the Swiss National Science Foundation (SNF) grant 310030_124970/1 (to A.B.), by a travel grant from the IIS-IP (to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). The CNIC is supported by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (Spanish Government award SEV-2015-0505). CIC biomaGUNE is supported by the María de Maeztu Units of Excellence Program from the Spanish Government (MDM-2017-0720). P.H.-A. was a recipient of a predoctoral FPU fellowship from the Spanish Government. E.N. is a recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM). A.M.-R., L.M. and J.E. are supported by the I3SNS or ‘Miguel Servet’ programmes (ISCIII, Spanish Government; partially funded by the FEDER/ERDF).

Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to ‘deactive’ form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na/H antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia., This research has been financed by Spanish Government grants (partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R.), SAF2015-71521-REDC (Consolredox network, to A.M.-R.), PI12/00875 and PI15/00107 (to A.M.-R), and SAF2013-32223 (to M.G.L.), by a grant from the Fundación Domingo Martínez (Ayuda a la Investigación 2014. Área de Biomedicina y Salud), by Swiss National Science Foundation (SNF) grant 310030_124970/1 to A.B, by a travel grant from the Instituto de Investigación Sanitaria Princesa (Ayuda para estancia breve 2012, to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). P.H.-A. and I.B. are recipients of predoctoral FPU fellowships (AP2010-1219 and AP2012-5621) from the Spanish Government, E.N. is recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM; FPI-UAM2012), and A.M.-R. and J.E. are supported by the I3SNS or “Miguel Servet” programmes (CES12/005 and CP14/00008; ISCIII, Spanish Government; partially funded by FEDER/ERDF).

et al., Oxygen is a key molecule for cell metabolism. Eukaryotic cells sense the reduction in oxygen availability (hypoxia) and trigger a series of cellular and systemic responses to adapt to hypoxia, including the optimization of oxygen consumption. Many of these responses are mediated by a genetic program induced by the hypoxia-inducible transcription factors (HIFs), regulated by a family of prolyl hydroxylases (PHD or EGLN) that use oxygen as a substrate producing HIF hydroxylation. In parallel to these oxygen sensors modulating gene expression within hours, acute modulation of protein function in response to hypoxia is known to occur within minutes. Free radicals acting as second messengers, and oxidative posttranslational modifications, have been implied in both groups of responses. Localization and speciation of the paradoxical increase in reactive oxygen species production in hypoxia remain debatable. We have observed that several cell types respond to acute hypoxia with a transient increase in superoxide production for about 10 min, probably originating in the mitochondria. This may explain in part the apparently divergent results found by various groups that have not taken into account the time frame of hypoxic ROS production. We propose that this acute and transient hypoxia-induced superoxide burst may be translated into oxidative signals contributing to hypoxic adaptation and preconditioning., This research has been financed by the Spanish government Grants CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 program; to S.L. and A.M.-R.), CP07/00143 (Miguel Servet program), PS09/00101 and PI12/00875 (to A.M.-R), and SAF2009-7520, SAF 2012-31338, and the “New Indigo” Partnership Program “Nitroxdiab” (PIM2010ENI-00631) (to S.L.); by Swiss National Science Foundation Grant 310030_124970/1 to A.B., by a travel grant from the Instituto de Investigación Sanitaria Princesa (to P.H.-A.); and by the COST actions TD0901 (HypoxiaNet) BM1005 (ENOG–European Network on Gasotransmitters) and BM1203 (EU-ROS). P.H.-A. is the recipient of an FPU fellowship from the Spanish government, and A.M.-R. is supported by the I3SNS program (ISCIII, Spanish government).