RESUMEN
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
Asunto(s)
Glutamato-Cisteína Ligasa , Glutatión , Animales , Ratones , Butionina Sulfoximina/farmacología , Modelos Animales de Enfermedad , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are linked to many diseases, including cancer and neurodegenerative disorders, determining the function of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus Thermophilus (GshF). GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes revealed metabolic liabilities under compartmentalized GSH depletion. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the role of GSH availability in physiology and disease.
RESUMEN
Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.
Asunto(s)
Proteasas ATP-Dependientes , ATPasas Asociadas con Actividades Celulares Diversas , Glutatión , Mitocondrias , Proteínas Mitocondriales , Proteínas de Transporte de Fosfato , Glutatión/metabolismo , Homeostasis , Hierro/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteómica , Retroalimentación Fisiológica , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Humanos , Proteínas Hierro-Azufre/metabolismo , Proteolisis , Células HEK293 , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismoRESUMEN
Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.
Asunto(s)
Glutatión/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Transporte Biológico , Proliferación Celular , Células Cultivadas , Eritropoyesis , Glutatión/deficiencia , Homeostasis , Humanos , Proteínas Hierro-Azufre/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/genética , Oxidación-Reducción , Proteoma , ProteómicaRESUMEN
Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.
Asunto(s)
Cistina/metabolismo , Ferroptosis/genética , Regulación Neoplásica de la Expresión Génica , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Tetrahidrofolato Deshidrogenasa/genética , Antineoplásicos/farmacología , Antioxidantes/farmacología , Biopterinas/análogos & derivados , Biopterinas/farmacología , Carbolinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cistina/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Ferroptosis/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Perfilación de la Expresión Génica , Humanos , Células Jurkat , Peroxidación de Lípido/efectos de los fármacos , Metotrexato/farmacología , Estrés Oxidativo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidores , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Piperazinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Tetrahidrofolato Deshidrogenasa/metabolismo , Vitamina E/farmacologíaRESUMEN
The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.
Asunto(s)
Proliferación Celular/fisiología , Hierro/metabolismo , Lisosomas/metabolismo , Colesterol/biosíntesis , Colesterol/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Células Jurkat , Lisosomas/fisiología , Mitocondrias/metabolismo , Transducción de Señal/genéticaRESUMEN
Cells can take up cysteine or synthesize it de novo from methionine, but synthesis alone does not meet the high demands of cancer cells to proliferate. In this issue, Zhu et al. (2019) identify the SAH:SAM ratio, indicative of the cellular methylation state, as limiting for effective cysteine synthesis and the growth of some tumors.
Asunto(s)
Cisteína , Neoplasias , Humanos , Metionina , MetilaciónRESUMEN
Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.
Asunto(s)
Axones/patología , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/fisiología , Animales , Axones/enzimología , Caspasa 3/fisiología , Células Cultivadas , Proteínas del Citoesqueleto , Activación Enzimática , Ganglios Espinales/citología , Ganglios Espinales/embriología , Ratones , Ratones Noqueados , Degeneración Nerviosa/enzimología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficiencia , Fosforilación , Procesamiento Proteico-Postraduccional , Receptor trkA/fisiología , Células Receptoras Sensoriales/fisiología , Relación Estructura-ActividadRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.