RESUMEN
The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
Asunto(s)
Dipéptidos , Lisosomas , Lisosomas/metabolismo , Animales , Dipéptidos/metabolismo , Oocitos/metabolismo , Microscopía por Crioelectrón , Ratones , Xenopus laevis , Humanos , Ratones Noqueados , Simulación de Dinámica Molecular , Simportadores/metabolismo , Simportadores/genética , Simportadores/química , Femenino , Canales de Potencial de Receptor TransitorioRESUMEN
A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.
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Técnicas de Cultivo de Célula , Ensayos Analíticos de Alto Rendimiento , Humanos , Línea Celular , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
During T cell activation, the transmembrane adaptor protein LAT (linker for activation of T cells) forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. LAT has also been associated with cholesterol-rich condensed lipid domains; However, the potential coupling between protein condensation and lipid phase separation and its role in organizing T cell signaling were unknown. Here, we report that LAT/Grb2/Sos1 condensates reconstituted on model membranes can induce and template lipid domains, indicating strong coupling between lipid- and protein-based phase separation. Correspondingly, activation of T cells induces cytoplasmic protein condensates that associate with and stabilize raft-like membrane domains. Inversely, lipid domains nucleate and stabilize LAT protein condensates in both reconstituted and living systems. This coupling of lipid and protein assembly is functionally important, as uncoupling of lipid domains from cytoplasmic protein condensates abrogates T cell activation. Thus, thermodynamic coupling between protein condensates and ordered lipid domains regulates the functional organization of living membranes.
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Proteínas de la Membrana , Linfocitos T , Linfocitos T/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , LípidosRESUMEN
A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.
RESUMEN
The composition of the plasma membrane (PM) must be tightly controlled despite constant, rapid endocytosis, which requires active, selective recycling of endocytosed membrane components. For many proteins, the mechanisms, pathways, and determinants of this PM recycling remain unknown. We report that association with ordered, lipid-driven membrane microdomains (known as rafts) is sufficient for PM localization of a subset of transmembrane proteins and that abrogation of raft association disrupts their trafficking and leads to degradation in lysosomes. Using orthogonal, genetically encoded probes with tunable raft partitioning, we screened for the trafficking machinery required for efficient recycling of engineered microdomain-associated cargo from endosomes to the PM. Using this screen, we identified the Rab3 family as an important mediator of PM localization of microdomain-associated proteins. Disruption of Rab3 reduced PM localization of raft probes and led to their accumulation in Rab7-positive endosomes, suggesting inefficient recycling. Abrogation of Rab3 function also mislocalized the endogenous raft-associated protein Linker for Activation of T cells (LAT), leading to its intracellular accumulation and reduced T cell activation. These findings reveal a key role for lipid-driven microdomains in endocytic traffic and suggest Rab3 as a mediator of microdomain recycling and PM composition.
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Endocitosis , Proteínas de la Membrana , Membrana Celular , Movimiento Celular , Lípidos , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.
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Ésteres , Glicerofosfolípidos , Fosfatos de Inositol , Lisosomas , Glicoproteínas de Membrana , Chaperonas Moleculares , Animales , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Niño , Ésteres/metabolismo , Glicerofosfolípidos/líquido cefalorraquídeo , Glicerofosfolípidos/metabolismo , Humanos , Fosfatos de Inositol/líquido cefalorraquídeo , Fosfatos de Inositol/metabolismo , Enfermedades por Almacenamiento Lisosomal/líquido cefalorraquídeo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Lisosomas/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/líquido cefalorraquídeo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismoRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.
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Dieta , Leucina , Hígado , Diana Mecanicista del Complejo 1 de la Rapamicina , Sestrinas , Tejido Adiposo Blanco/enzimología , Animales , Leucina/metabolismo , Hígado/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Sestrinas/metabolismo , Transducción de SeñalRESUMEN
Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome-the organelle, related to the lysosome, that synthesizes pigment-but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine-the oxidized dimer of cysteine-in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7-9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.
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Cisteína/metabolismo , Lisosomas/metabolismo , Melanosomas/metabolismo , Proteínas de la Membrana/metabolismo , Transporte Biológico , Fraccionamiento Celular , Línea Celular , Cistina/metabolismo , Cistinosis/genética , Cistinosis/metabolismo , Fibroblastos , Humanos , Melaninas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Oxidación-ReducciónRESUMEN
The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as coessential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.
RESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.
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Dihidroxiacetona Fosfato/fisiología , Glucosa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Dihidroxiacetona/metabolismo , Dihidroxiacetona Fosfato/biosíntesis , Metabolismo Energético , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa/deficiencia , Glucólisis , Células HEK293 , Humanos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Increased glucose uptake and metabolism is a prominent phenotype of most cancers, but efforts to clinically target this metabolic alteration have been challenging. Here, we present evidence that lactoylglutathione (LGSH), a byproduct of methylglyoxal detoxification, is elevated in both human and murine non-small cell lung cancers (NSCLC). Methylglyoxal is a reactive metabolite byproduct of glycolysis that reacts non-enzymatically with nucleophiles in cells, including basic amino acids, and reduces cellular fitness. Detoxification of methylglyoxal requires reduced glutathione (GSH), which accumulates to high levels in NSCLC relative to normal lung. Ablation of the methylglyoxal detoxification enzyme glyoxalase I (Glo1) potentiates methylglyoxal sensitivity and reduces tumor growth in mice, arguing that targeting pathways involved in detoxification of reactive metabolites is an approach to exploit the consequences of increased glucose metabolism in cancer.
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Glucosa/metabolismo , Glucólisis , Neoplasias Pulmonares/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Glutatión/metabolismo , Humanos , Inactivación Metabólica , Lactoilglutatión Liasa/metabolismo , Pulmón/metabolismo , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Piruvaldehído/metabolismo , Piruvaldehído/toxicidadRESUMEN
Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (m6A) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3' UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1. Transcriptome-wide, RME1 is the primary message that displays both increased methylation and reduced expression in an Ime4p-dependent manner. In yeast strains for which IME4 is dispensable for meiosis, a natural polymorphism in the RME1 promoter reduces RME1 transcription, obviating the requirement for methylation. Mutation of a single m6A site in the RME1 3' UTR increases Rme1p repressor production and reduces meiotic efficiency. These results reveal the molecular and physiological consequences of a modification in the 3' UTR of an mRNA.
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Regiones no Traducidas 3' , Adenosina/análogos & derivados , ARN Mensajero/genética , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Regulación Fúngica de la Expresión Génica , Meiosis , Metilación , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Glutatión/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Sistemas de Translocación de Proteínas/metabolismo , Línea Celular Tumoral , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Matriz Extracelular/metabolismo , Humanos , Fosfogluconato Deshidrogenasa/fisiología , Sistemas de Translocación de Proteínas/fisiologíaRESUMEN
Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.
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Líquido Extracelular/química , Neoplasias/patología , Nutrientes/análisis , Microambiente Tumoral , Animales , Línea Celular Tumoral , Femenino , Xenoinjertos/patología , Masculino , Metabolómica , Ratones Endogámicos C57BL , Plasma/químicaRESUMEN
The committed step in DNA replication initiation is the activation of the Mcm2-7 replicative DNA helicase. Two activators, Cdc45 and GINS, associate with Mcm2-7 at origins of replication to form the CMG complex, which is the active eukaryotic replicative helicase. These activators function during both replication initiation and elongation, however, it remains unclear whether Cdc45 performs the same function(s) during both events. Here, we describe the genetic and biochemical characterization of seven Cdc45 mutations. Three of these mutations are temperature-sensitive lethal mutations in CDC45. Intriguingly, these mutants are defective for DNA replication initiation but not elongation. Consistent with an initiation defect, all three temperature-sensitive mutants are defective for CMG formation. Two of the lethal mutants are located within the RecJ-like domain of Cdc45 confirming the importance of this region for Cdc45 function. The remaining two lethal mutations localize to an intrinsically disordered region (IDR) of Cdc45 that is found in all eukaryotes. Despite the lethality of these IDR substitution mutants, Cdc45 lacking the IDR retains full function. Together, our data provide insights into the functional importance of Cdc45 domains and suggest that the requirements for Cdc45 function during DNA replication initiation are distinct from those involved in replication elongation.
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Alelos , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Nucleares/química , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , TemperaturaRESUMEN
Cholesterol is essential for cells to grow and proliferate. Normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis1, but the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identify a subset of cancer cells that is auxotrophic for cholesterol and thus highly dependent on its uptake. Through metabolic gene expression analysis, we pinpoint the loss of squalene monooxygenase expression as a cause of cholesterol auxotrophy, particularly in ALK+ anaplastic large cell lymphoma (ALCL) cell lines and primary tumours. Squalene monooxygenase catalyses the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK+ ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumour xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability and, more broadly, that systematic approaches can be used to identify nutrient dependencies unique to individual cancer types.
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Apoptosis , Colesterol/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Estrés Oxidativo , Escualeno/metabolismo , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular , Colesterol/biosíntesis , Código de Barras del ADN Taxonómico , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/metabolismo , Femenino , Humanos , Hierro/metabolismo , Linfoma Anaplásico de Células Grandes/enzimología , Masculino , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Receptores de LDL/genética , Receptores de LDL/metabolismo , Escualeno-Monooxigenasa/genética , Escualeno-Monooxigenasa/metabolismo , Adulto JovenRESUMEN
Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.
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Epítopos/inmunología , Mitocondrias/inmunología , Animales , Hepatocitos/metabolismo , Immunoblotting , Lípidos/fisiología , Masculino , Metabolómica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/química , Mitocondrias/metabolismo , Mitocondrias/fisiología , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/inmunología , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/fisiología , Proteómica/métodosRESUMEN
One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.
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Mitocondrias/metabolismo , Serina/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas , Carbono/metabolismo , Pruebas Genéticas , Humanos , Células Jurkat , Células K562 , Transportador 1 de Sodio-Glucosa/genéticaRESUMEN
The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.