RESUMO
Iron is the most abundant transition metal essential for numerous cellular processes. Although most mammalian cells acquire iron through transferrin receptors, molecular players of iron utilization under iron restriction are incompletely understood. To address this, we performed metabolism-focused CRISPRa gain-of-function screens, which revealed metabolic limitations under stress conditions. Iron restriction screens identified not only expected members of iron utilization pathways but also SLCO2B1, a poorly characterized membrane carrier. SLCO2B1 expression is sufficient to increase intracellular iron, bypass the essentiality of the transferrin receptor, and enable proliferation under iron restriction. Mechanistically, SLCO2B1 mediates heme analog import in cellular assays. Heme uptake by SLCO2B1 provides sufficient iron for proliferation through heme oxygenases. Notably, SLCO2B1 is predominantly expressed in microglia in the brain, and primary Slco2b1-/- mouse microglia exhibit strong defects in heme analog import. Altogether, our work identifies SLCO2B1 as a microglia-enriched plasma membrane heme importer and provides a genetic platform to identify metabolic limitations under stress conditions.
Assuntos
Heme , Ferro , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico , Heme/genética , Heme/metabolismo , Ferro/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Ativação TranscricionalRESUMO
Pigmented epithelioid melanocytoma (PEM) is a unique tumor with significantly pigmented appearance and indolent behavior; however, it can demonstrate cytological atypia and metastasize to local lymph nodes. Clinical and histomorphological overlap between PEM and its lower or higher-grade mimics can make it difficult to distinguish in certain cases. Genomic, transcriptomic and epigenetic data indicate that PEMs are molecularly distinct entities from other melanocytic neoplasms and melanomas. In addition, methylation studies are emerging as a tool that can be useful in difficult cases. In this review, we focus on the clinical, histopathologic and recent insights in the molecular features of pigmented epithelioid melanocytic melanocytomas and their mimics. We also present a challenging case that was resolved using methylation analysis providing a proof of concept for using epigenetic studies for similar challenging cases.
RESUMO
Coessentiality mapping has been useful to systematically cluster genes into biological pathways and identify gene functions1-3. Here, using the debiased sparse partial correlation (DSPC) method3, we construct a functional coessentiality map for cellular metabolic processes across human cancer cell lines. This analysis reveals 35 modules associated with known metabolic pathways and further assigns metabolic functions to unknown genes. In particular, we identify C12orf49 as an essential regulator of cholesterol and fatty acid metabolism in mammalian cells. Mechanistically, C12orf49 localizes to the Golgi, binds membrane-bound transcription factor peptidase, site 1 (MBTPS1, site 1 protease) and is necessary for the cleavage of its substrates, including sterol regulatory element binding protein (SREBP) transcription factors. This function depends on the evolutionarily conserved uncharacterized domain (DUF2054) and promotes cell proliferation under cholesterol depletion. Notably, c12orf49 depletion in zebrafish blocks dietary lipid clearance in vivo, mimicking the phenotype of mbtps1 mutants. Finally, in an electronic health record (EHR)-linked DNA biobank, C12orf49 is associated with hyperlipidaemia through phenome analysis. Altogether, our findings reveal a conserved role for C12orf49 in cholesterol and lipid homeostasis and provide a platform to identify unknown components of other metabolic pathways.
Assuntos
Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Hiperlipidemias/genética , Metabolismo dos Lipídeos/genética , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Peixe-ZebraRESUMO
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.
Assuntos
Proliferação de Células/fisiologia , Ferro/metabolismo , Lisossomos/metabolismo , Colesterol/biossíntese , Colesterol/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Células Jurkat , Lisossomos/fisiologia , Mitocôndrias/metabolismo , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Epitopos/imunologia , Mitocôndrias/imunologia , Animais , Hepatócitos/metabolismo , Immunoblotting , Lipídeos/fisiologia , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias Hepáticas/química , Mitocôndrias Hepáticas/imunologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Proteômica/métodosRESUMO
Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Glicina Hidroximetiltransferase/metabolismo , Mitocôndrias/genética , Iniciação Traducional da Cadeia Peptídica , RNA de Transferência de Metionina/química , Serina/química , Animais , Apoptose , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Citosol/metabolismo , Feminino , Glicina Hidroximetiltransferase/antagonistas & inibidores , Glicina Hidroximetiltransferase/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Serina/genética , Serina/metabolismo , Tetra-Hidrofolatos/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human ρ° cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.
Assuntos
Mitocôndrias/enzimologia , Proteínas/antagonistas & inibidores , Antimicina A/análogos & derivados , Antimicina A/farmacologia , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Fosforilação Oxidativa , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteínas/metabolismo , Proteína Inibidora de ATPaseRESUMO
As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.