RESUMEN
In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.
Asunto(s)
Retículo Endoplásmico/enzimología , Proteínas Fúngicas/metabolismo , Glutatión/metabolismo , Glicoproteínas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Canales de Translocación SEC/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/enzimología , Difusión Facilitada , Proteínas Fúngicas/genética , Disulfuro de Glutatión/metabolismo , Glicoproteínas/genética , Proteínas HSP70 de Choque Térmico/genética , Peróxido de Hidrógeno/metabolismo , Membranas Intracelulares/enzimología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Canales de Translocación SEC/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Tiempo , Respuesta de Proteína DesplegadaRESUMEN
Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.
Asunto(s)
Adenosina Trifosfato/análisis , Metabolismo Energético/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Análisis de la Célula Individual/métodos , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1). Previous studies in yeast demonstrate that the yeast GSH/Grx (glutaredoxin) redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. In the present study, we probe further the interaction between hSOD1, GSH and Grxs to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that hGrx1 (human Grx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT (wild-type) hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared with WT hSOD1. Furthermore, the presence of metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT compared with ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Glutarredoxinas/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Disulfuros/química , Glutarredoxinas/química , Glutarredoxinas/genética , Humanos , Cinética , Mutación , Oxidación-Reducción , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , TermodinámicaRESUMEN
Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.