RESUMEN
The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NOâ) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana , NADPH Oxidasa 2 , Oxidación-Reducción , Especies Reactivas de Oxígeno , Transducción de Señal , Proteína de Unión al GTP rac1 , Humanos , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico/metabolismo , Movimiento Celular , Fosforilación , Senescencia Celular , Técnicas de Silenciamiento del GenRESUMEN
Chemogenetics refers to experimental methods that use novel recombinant proteins that can be dynamically and uniquely regulated by specific biochemicals. Chemogenetic approaches allow the precise manipulation of cellular signaling to delineate the molecular pathways involved in both physiological and pathological disease states. Approaches utilizing yeast d-amino acid oxidase (DAAO) enable manipulation of intracellular redox metabolism through generation of hydrogen peroxide in the presence of d-amino acids and have led to the development of new and informative animal models to characterize the impact of oxidative stress in heart failure and neurodegeneration. These chemogenetic models, in which DAAO expression is regulated by different tissue-specific promoters, have led to a range of cardiac phenotypes. This review discusses chemogenetic approaches to manipulate oxidative stress in models of heart failure. These approaches provide new insights into the relationships between redox metabolism and normal and pathologic states in the heart, as well as in other diseases characterized by oxidative stress.
Asunto(s)
Insuficiencia Cardíaca , Animales , Oxidación-Reducción , Insuficiencia Cardíaca/genética , Estrés Oxidativo , AminoácidosRESUMEN
Chemogenetic approaches have been developed to define the mechanisms whereby the intracellular oxidant hydrogen peroxide (H2O2) modulates both physiological and pathological responses. Recombinant yeast D-amino acid oxidase (DAAO) can be exploited to modulate H2O2 in target cells and tissues. In vitro studies using cultured cells expressing recombinant DAAO have provided critical new information on the intracellular transport and metabolism of H2O2 with great temporal and spatial resolution. In contrast, in vivo studies using chemogenetic/transgenic animal models have explored the pathological effects of chronically elevated H2O2 in tissues. Coupled with transcriptomic, proteomic, and metabolomic methods, in vivo chemogenetic approaches are providing new insights into the adaptations to oxidative stress. This review of chemogenetic applications focuses on new models of heart failure and neurodegeneration that leverage in vivo chemogenetic modulation of oxidative stress in target tissues to identify new therapeutic targets.