RESUMO
The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.
Assuntos
Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Animais , Proteínas de Transporte , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Genes Essenciais , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Mitocondriais , Processamento de Proteína Pós-Traducional , Proteínas ras/genéticaRESUMO
The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos Essenciais/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Arginina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Alinhamento de SequênciaRESUMO
Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles.
Assuntos
Redes e Vias Metabólicas , Metaboloma , Mitocôndrias/metabolismo , Transporte de Elétrons/genética , Células HeLa , Humanos , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologiaRESUMO
The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.
Assuntos
Ácido Aspártico/biossíntese , Proliferação de Células , Transporte de Elétrons , Mitocôndrias/metabolismo , Aspartato Aminotransferase Citoplasmática/metabolismo , Ácido Aspártico/metabolismo , DNA Mitocondrial/genética , Humanos , Células Jurkat , Mutação , Fenformin/farmacologia , Ácido Pirúvico/metabolismoRESUMO
It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.
Assuntos
Transição Epitelial-Mesenquimal , Pirimidinas/metabolismo , Animais , Carcinoma/metabolismo , Linhagem Celular Tumoral , Di-Hidrouracila Desidrogenase (NADP)/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
Hyperkalaemia is an electrolyte imbalance that impairs muscle function and myocardial excitability, and can potentially lead to fatal arrhythmias and sudden cardiac death. The prevalence of hyperkalaemia is estimated to be 6%-7% worldwide and 7%-10% in Asia. Hyperkalaemia frequently affects patients with chronic kidney disease, heart failure, and diabetes mellitus, particularly those receiving treatment with renin-angiotensin-aldosterone system (RAAS) inhibitors. Both hyperkalaemia and interruption of RAAS inhibitor therapy are associated with increased risks for cardiovascular events, hospitalisations, and death, highlighting a clinical dilemma in high-risk patients. Conventional potassium-binding resins are widely used for the treatment of hyperkalaemia; however, caveats such as the unpalatable taste and the risk of gastrointestinal side effects limit their chronic use. Recent evidence suggests that, with a rapid onset of action and improved gastrointestinal tolerability, novel oral potassium binders (e.g., patiromer and sodium zirconium cyclosilicate) are alternative treatment options for both acute and chronic hyperkalaemia. To optimise the care for patients with hyperkalaemia in the Asia-Pacific region, a multidisciplinary expert panel was convened to review published literature, share clinical experiences, and ultimately formulate 25 consensus statements, covering three clinical areas: (i) risk factors of hyperkalaemia and risk stratification in susceptible patients; (ii) prevention of hyperkalaemia for at-risk individuals; and (iii) correction of hyperkalaemia for at-risk individuals with cardiorenal disease. These statements were expected to serve as useful guidance in the management of hyperkalaemia for health care providers in the region.
Assuntos
Consenso , Hiperpotassemia , Humanos , Hiperpotassemia/epidemiologia , Hiperpotassemia/terapia , Hiperpotassemia/diagnóstico , Ásia/epidemiologia , Fatores de Risco , Potássio/sangue , Silicatos/uso terapêutico , Silicatos/efeitos adversosRESUMO
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
Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Glicina Hidroximetiltransferase/metabolismo , Glicina/metabolismo , Isquemia/metabolismo , Acetona/análogos & derivados , Acetona/metabolismo , Acetona/toxicidade , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/enzimologia , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/enzimologia , Glicina Desidrogenase (Descarboxilante)/antagonistas & inibidores , Glicina Desidrogenase (Descarboxilante)/metabolismo , Humanos , Isquemia/enzimologia , Isquemia/patologia , Camundongos , Necrose , Consumo de Oxigênio , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/toxicidade , Piruvato Quinase/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Biguanidas/farmacologia , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Glucose/deficiência , Neoplasias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Tipagem Molecular , Mutação , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Fenformin/farmacologia , Interferência de RNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Recently, new evidence from large scale trials and updated guidelines have emerged on the risks and benefits of extended dual antiplatelet therapy (DAPT) for patients with acute coronary syndrome (ACS). AIMS: To discuss, clarify and advise on the application of the evidences and guidelines on individual patient selection for extended DAPT, with regard to balancing risk factors, particularly in Asian populations. METHODS: A total of 14 local cardiologists from Hong Kong with extensive experience in cardiology and cardiac interventions convened in a series of 3 advisory board meetings from October 2016 to September 2017, which included reviews of new evidence in the literature and discussions of the latest clinical trends, using an anonymous, electronic voting system for arriving at consensuses. RESULTS: Recommendations were produced for the following nine risk factors: old age (>65), chronic kidney disease (CKD), diabetes mellitus (DM), recurrent myocardial infarction (MI), multi-vessel disease (MVD), multiple stents, bioresorbable vascular scaffold (BVS) stent, left main stenting and peripheral artery disease (PAD). Strong ischaemic risk factors include DM, recurrent MI, MVD and/or >3 stents; less-strong ischaemic factors include CKD, left main stenting, BVS stent and PAD. Old age can be an unclear risk factor due to variations in physical fitness even among patients of identical age. DISCUSSION: The strengths and limitations of the results were acknowledged. CONCLUSION: ACS patients with ischaemic risk factors could be considered for extended DAPT beyond 12 months, while balancing the risk of bleeding.
Assuntos
Síndrome Coronariana Aguda/terapia , Stents Farmacológicos/efeitos adversos , Inibidores da Agregação Plaquetária/administração & dosagem , Guias de Prática Clínica como Assunto , Síndrome Coronariana Aguda/diagnóstico , Comitês Consultivos , Esquema de Medicação , Quimioterapia Combinada , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Hong Kong , Humanos , Infarto do Miocárdio/etiologia , Inibidores da Agregação Plaquetária/efeitos adversos , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Fatores de Risco , Trombose/etiologia , Trombose/prevenção & controleRESUMO
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
Assuntos
Carbono/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Serina/biossíntese , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Carbono/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Feminino , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Estrutura Molecular , Fosfoglicerato Desidrogenase/metabolismo , Purinas/biossíntese , Serina/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Timidina/biossíntese , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Genômica , Serina/biossíntese , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclo do Ácido Cítrico/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Melanoma/enzimologia , Melanoma/genética , Camundongos , Transplante de Neoplasias , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Interferência de RNARESUMO
Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.
RESUMO
ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1RESUMO
Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in â¼12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.
Assuntos
Separação Imunomagnética/métodos , Metaboloma/fisiologia , Metabolômica/métodos , Mitocôndrias/química , Mitocôndrias/metabolismo , Cromatografia Líquida , Técnicas Citológicas , Células HeLa , Humanos , Espectrometria de MassasRESUMO
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
The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.
Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Lisossomos/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neoplasias/enzimologia , Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Mutação , Neoplasias/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA-resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.
Assuntos
Transportadores de Ácidos Monocarboxílicos/genética , Piruvatos/metabolismo , Simportadores/genética , Animais , Transporte Biológico/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Camundongos , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/metabolismo , Piruvatos/farmacologia , Simportadores/metabolismoRESUMO
Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013.