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BACKGROUND AIMS: Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. METHODS: To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. RESULTS: We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. CONCLUSIONS: These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome.
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Proliferación Celular , Glucólisis , Inmunoterapia Adoptiva , Fosforilación Oxidativa , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Melanoma/terapia , Melanoma/inmunología , Activación de Linfocitos , Citotoxicidad Inmunológica , Línea Celular Tumoral , Microambiente TumoralRESUMEN
In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.
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Rotura Cromosómica , Sitios Frágiles del Cromosoma/genética , Dosificación de Gen/genética , Longevidad/genética , Proteínas Serina-Treonina Quinasas/genética , Ribonucleósido Difosfato Reductasa/genética , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Activación Enzimática/genética , Fibroblastos/citología , Humanos , Ratones , Nucleósidos/metabolismo , Análisis de SupervivenciaRESUMEN
In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.
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ADN Helicasas/genética , Replicación del ADN/genética , ADN/biosíntesis , Complejo de Reconocimiento del Origen/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , División Celular/genética , Cromatina/genética , ADN/genética , Daño del ADN/genética , ADN-Topoisomerasas/genética , Proteínas de Unión al ADN/genética , Mutación Puntual , Origen de Réplica/genética , Puntos de Control de la Fase S del Ciclo Celular/genética , Saccharomyces cerevisiae/genéticaRESUMEN
As up to a fifth of all cancers worldwide, have now been linked to microbial infections, it is essential to understand the carcinogenic nature of the bacterial/host interaction. This paper reviews the bacterial targeting of mediators of mitochondrial genomic fidelity and of mitochondrial apoptotic pathways, and compares the impact of the bacterial alteration of mitochondrial function to that of cancer. Bacterial virulence factors have been demonstrated to induce mutations of mitochondrial DNA (mtDNA) and to modulate DNA repair pathways of the mitochondria. Furthermore, virulence factors can induce or impair the intrinsic apoptotic pathway. The effect of bacterial targeting of mitochondria is analogous to behavior of mitochondria in a wide array of tumours, and this strongly suggests that mitochondrial targeting of bacteria is a risk factor for carcinogenesis.
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Infecciones Bacterianas/complicaciones , Carcinogénesis , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Apoptosis , Carcinogénesis/genética , Carcinogénesis/metabolismo , Daño del ADN , Metabolismo Energético , Humanos , Mutación , Transducción de SeñalRESUMEN
Mitophagy is a mechanism that maintains mitochondrial integrity and homeostasis and is thought to promote longevity and reduce the risk of age-related neurodegenerative diseases, including Alzheimer's disease (AD). Here, we investigate the abundance of mitochondrial reactive oxygen species (ROS), mitochondrial function, and mitophagy in primary fibroblasts from patients with sporadic AD (sAD) and normal healthy controls. The results show increased levels of mitochondrial ROS, changes in mitochondrial morphology, altered bioenergetic properties, and defects in autophagy, mitophagy, and lysosome-mediated degradation pathways in sAD fibroblasts relative to control fibroblasts. Interestingly, lysosome abundance and the staining of lysosomal markers remained high, while the capacity of lysosome-dependent degradation was lower in sAD fibroblasts than in controls fibroblasts. Nicotinamide riboside supplementation decreased mitochondrial ROS, while capacity for lysosomal degradation remained unchanged in sAD fibroblasts relative to healthy control fibroblasts. These findings provide insight into molecular mechanisms involving the dysregulation of lysosome and autophagy/mitophagy pathways that may contribute significantly to clinical signs and pathological features of sAD.
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Enfermedad de Alzheimer , Autofagia , Fibroblastos , Lisosomas , Mitocondrias , Mitofagia , Especies Reactivas de Oxígeno , Humanos , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Lisosomas/metabolismo , Lisosomas/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Especies Reactivas de Oxígeno/metabolismo , Femenino , Masculino , Anciano , Persona de Mediana Edad , Anciano de 80 o más Años , Células Cultivadas , Estudios de Casos y Controles , Niacinamida/análogos & derivados , Niacinamida/farmacología , Compuestos de PiridinioRESUMEN
Nucleotide metabolism plays a crucial role in the regulation of the tumor microenvironment (TME) and immune cell function. In the TME, limited availability of nucleotide precursors due to increased consumption by tumor cells and T cells affects both tumor development and immune function. Metabolic reprogramming in tumor cells favors pathways supporting growth and proliferation, including nucleotide synthesis. Additionally, extracellular nucleotides, such as ATP and adenosine, exhibit dual roles in modulating immune function and tumor cell survival. ATP stimulates antitumor immunity by activating purinergic receptors, while adenosine acts as a potent immunosuppressor. Targeting nucleotide metabolism in the TME holds immense promise for cancer therapy. Understanding the intricate relationship between nucleotide metabolism, the TME, and immune responses will pave the way for innovative therapeutic interventions.
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Neoplasias , Microambiente Tumoral , Humanos , Neoplasias/terapia , Adenosina , Adenosina Trifosfato/metabolismo , NucleótidosRESUMEN
FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.
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Síndrome de Rett , Humanos , Encéfalo/metabolismo , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Factores de Transcripción Forkhead/genética , Proteínas del Tejido NerviosoRESUMEN
Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1-/- mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1-/- MEFs can be partially reversed by NR supplementation, which also protects the mitochondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs.
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Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine-DNA glycosylase or oxoguanine DNA glycosylase (hOGG1) sensitive sites, in mono-cultures of A549 or THP-1a and co-cultures of A549 and THP-1a cells. The strongest genotoxic effects were observed in A549 mono-cultures and SRM2975 was more potent than SRM1650b. The ROS production only increased in cells exposed to SRM2975, with strongest concentration-dependent effect in the THP-1a mono-cultures. The basal respiration level in THP-1a cells increased on exposure to SRM1650b and SRM2975 without indication of mitochondrial dysfunction. This is consistent with activation of the cells and there was no direct relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells.
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Daño del ADN/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Macrófagos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Línea Celular Tumoral , Técnicas de Cocultivo , Ensayo Cometa , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN-Formamidopirimidina Glicosilasa , Células Epiteliales/citología , Células Epiteliales/patología , Humanos , Inflamación/patología , Pulmón/citología , Pulmón/patología , Macrófagos/citología , Macrófagos/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The correlation between mitochondrial function and oncogenesis is complex and is not fully understood. Here we determine the importance of mitochondrial-linked pyrimidine synthesis for the aggressiveness of cancer cells. The enzyme dihydroorotate dehydrogenase (DHODH) links oxidative phosphorylation to de novo synthesis of pyrimidines. We demonstrate that an inhibition of DHODH results in a respiration-independent significant increase of anchorage-independent growth but does not affect DNA repair ability. Instead, we show an autophagy-independent increase of lysosomes. The results of this study suggest that inhibition of mitochondrial-linked pyrimidine synthesis in cancer cells results in a more aggressive tumor phenotype.
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Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Carcinogénesis , Dihidroorotato Deshidrogenasa , Humanos , Lisosomas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , PirimidinasRESUMEN
Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.
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Síndrome de Bloom/enzimología , Síndrome de Bloom/patología , Mitocondrias/metabolismo , Estrés Oxidativo , RecQ Helicasas/deficiencia , Autofagia , Ciclina B1/metabolismo , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Metabolismo Energético , Fibroblastos/enzimología , Fibroblastos/patología , Fase G1 , Humanos , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Mitosis , Especies Reactivas de Oxígeno/metabolismo , RecQ Helicasas/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.
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Activación de Linfocitos/inmunología , Mitocondrias/metabolismo , Pirimidinas/biosíntesis , Proliferación Celular/fisiología , Dihidroorotato Deshidrogenasa/antagonistas & inhibidores , Humanos , Mitocondrias/efectos de los fármacosRESUMEN
During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.
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Citocinas , Mitocondrias , Respiración de la Célula , Citocinas/metabolismo , Humanos , Mitocondrias/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , RespiraciónRESUMEN
Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.
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Atorvastatina/farmacología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/patología , Mitocondrias Musculares/metabolismo , Obesidad/patología , Animales , Biomarcadores/metabolismo , Respiración de la Célula , Citrato (si)-Sintasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Metaboloma , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Porcinos , Porcinos Enanos , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Background: Gilles de la Tourette syndrome (GTS) is a neurodevelopmental condition characterized by motor and vocal tics. The underlying etiology remains largely unknown, and GTS is considered as a complex multifactorial disorder associated with effects of several genes in combination with environmental factors. The inner mitochondrial membrane peptidase, subunit 2 (IMMP2L) has been suggested as one of the susceptibility genes for GTS, and IMMP2L-deficient mouse and human cells show increased levels of mitochondrial oxidative stress and altered cell fate programming. Hence, a potential involvement of IMMP2L-induced mitochondrial dysfunction in GTS pathology is yet to be elucidated. To address this, we investigated mitochondrial function in a group of GTS patients with intragenic IMMP2L deletions and compared with GTS without IMMP2L deletions and healthy controls. Methods: Mitochondrial function in fibroblasts from GTS patients and non-GTS parents (with and without IMMP2L deletions) compared to healthy controls were evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial mass, and mitochondrial respiration. In addition, we evaluated apoptosis and senescence. Results: None of the mitochondrial parameters assessed in this study were significantly distinctive when comparing GTS patients with and without IMMP2L deletions against healthy controls or parents with or without IMMP2L deletions, and we did not observe altered cell programming. Conclusion: This study suggests that IMMP2L deletions do not lead to a substantial general mitochondrial dysfunction in GTS fibroblasts. Assessing a large cohort of controls and patients of similar age and gender would possibly reveal small differences in mitochondrial function. However, it is possible that IMMP2L variants affect mitochondrial function during specific instances of stress stimuli or in brain regions suggested to be affected in GTS.
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Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology.
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Statins are prescribed to treat hypercholesterolemia and to reduce the risk of cardiovascular disease. However, statin users frequently report myalgia, which can discourage physical activity or cause patients to discontinue statin use, negating the potential benefit of the treatment. Although a proposed mechanism responsible for Statin-Associated Myopathy (SAM) suggests a correlation with impairment of mitochondrial function, the relationship is still poorly understood. Here, we provide evidence that long-term treatment of hypercholesterolemic patients with Simvastatin at a therapeutic dose significantly display increased mitochondrial respiration in peripheral blood mononuclear cells (PBMCs), and platelets compared to untreated controls. Furthermore, the amount of superoxide is higher in mitochondria in PBMCs, and platelets from Simvastatin-treated patients than in untreated controls, and the abundance of mitochondrial superoxide, but not mitochondrial respiration trends with patient-reported myalgia. Ubiquinone (also known as coenzyme Q10) has been suggested as a potential treatment for SAM; however, an 8-week course of oral ubiquinone had no impact on mitochondrial functions or the abundance of superoxide in mitochondria from PBMCs, and platelets. These results demonstrate that long-term treatment with Simvastatin increases respiration and the production of superoxide in mitochondria of PBMCs and platelets.
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Plaquetas/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipercolesterolemia/tratamiento farmacológico , Leucocitos Mononucleares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Simvastatina/farmacología , Plaquetas/metabolismo , Línea Celular , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipercolesterolemia/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Simvastatina/uso terapéutico , Superóxidos/metabolismoRESUMEN
Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition.
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Ácido Cítrico/metabolismo , Citoplasma/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunomodulación , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Femenino , Edición Génica , Regulación de la Expresión Génica , Glucólisis , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ligandos , Activación de Linfocitos , Linfocitos/inmunología , Linfocitos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Sitio de Iniciación de la TranscripciónRESUMEN
BACKGROUND: The definition of a hypothetical protein is a protein that is predicted to be expressed from an open reading frame, but for which there is no experimental evidence of translation. Hypothetical proteins constitute a substantial fraction of proteomes of human as well as of other eukaryotes. With the general belief that the majority of hypothetical proteins are the product of pseudogenes, it is essential to have a tool with the ability of pinpointing the minority of hypothetical proteins with a high probability of being expressed. RESULTS: Here, we present an in silico selection strategy where eukaryotic hypothetical proteins are sorted according to two criteria that can be reliably identified in silico: the presence of subcellular targeting signals and presence of characterized protein domains. To validate the selection strategy we applied it on a database of human hypothetical proteins dating to 2006 and compared the proteins predicted to be expressed by our selecting strategy, with their status in 2008. For the comparison we focused on mitochondrial proteins, since considerable amounts of research have focused on this field in between 2006 and 2008. Therefore, many proteins, defined as hypothetical in 2006, have later been characterized as mitochondrial. CONCLUSION: Among the total amount of human proteins hypothetical in 2006, 21% have later been experimentally characterized and 6% of those have been shown to have a role in a mitochondrial context. In contrast, among the selected hypothetical proteins from the 2006 dataset, predicted by our strategy to have a mitochondrial role, 53-62% have later been experimentally characterized, and 85% of these have actually been assigned a role in mitochondria by 2008.Therefore our in silico selection strategy can be used to select the most promising candidates for subsequent in vitro and in vivo analyses.
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Biología Computacional/métodos , Proteínas/química , Bases de Datos de Proteínas , Humanos , Sistemas de Lectura Abierta , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Defects in mitochondrial oxidative phosphorylation are a feature of many human diseases. To date, determination of oxidative phosphorylation has required fresh and live sample material and therefore also access to specialized equipment and trained personnel. Cryopreservation of samples is an attractive alternative, where samples can be collected and stored in an economic and practical fashion for later bulk assays. Here, we present an accurate, reliable method for estimating mitochondrial oxidative phosphorylation capacity of cryopreserved human cells. Broad adoption of this method will allow uncomplicated collection of samples and measurements of oxidative phosphorylation.