RESUMO
Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.
Assuntos
Doenças Autoimunes/metabolismo , Autoimunidade , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/patologia , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismoRESUMO
T-lymphocytes show large changes in ATP demand and nutrient utilization, imposed by their different roles as T memory and T effector cells. Therefore, T cell remodeling represents a bioenergetic challenge to mitochondria. New work from Buck et al. links changes in mitochondrial shape to T cell fate choice.
Assuntos
Mitocôndrias/metabolismo , Linfócitos T , Linfócitos T CD8-Positivos , Metabolismo Energético , Humanos , Memória ImunológicaRESUMO
Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Metabolismo Energético , Neoplasias Pulmonares , Mitocôndrias , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Ácidos Graxos/metabolismo , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Fenótipo , Tomografia por Emissão de PósitronsRESUMO
The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.
Assuntos
Trifosfato de Adenosina , Mitocôndrias , Camundongos , Animais , Trifosfato de Adenosina/metabolismo , Mitocôndrias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas/metabolismo , Homeostase , HidróliseRESUMO
Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet ß-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.
Assuntos
Ácidos Graxos , Malonil Coenzima A , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Malonil Coenzima A/farmacologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Oxirredução , Mitocôndrias/metabolismoRESUMO
To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1ß, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1ß or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.
Assuntos
Mitocôndrias , Telomerase/antagonistas & inibidores , Homeostase do Telômero , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Genes cdc , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Camundongos , Mitocôndrias/metabolismo , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Telomerase/genética , Telomerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and branched-chain α-ketoacid levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here, we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show that BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly sixfold less potent than the prototypical uncoupler 2,4-dinitrophenol and phenocopies 2,4-dinitrophenol in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest that the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.
Assuntos
Lipogênese , Doenças Metabólicas , Membranas Mitocondriais , Inibidores de Proteínas Quinases , Espécies Reativas de Oxigênio , Humanos , 2,4-Dinitrofenol/farmacologia , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Lipogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Camundongos , Ratos , Linhagem Celular , Membranas Mitocondriais/efeitos dos fármacos , Células CultivadasRESUMO
Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
Assuntos
Smegmamorpha , Animais , Smegmamorpha/metabolismo , Mitocôndrias/metabolismo , Metabolismo Energético , Glicólise , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Mamíferos/metabolismoRESUMO
SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.
Assuntos
Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sirtuínas/metabolismo , Animais , Respiração Celular , Transportador de Glucose Tipo 1 , Glicólise , Camundongos , Camundongos Knockout , Sirtuínas/genéticaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
Assuntos
Criopreservação , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Masculino , CamundongosRESUMO
The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Mioblastos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Cultivadas , Feminino , Células HeLa , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mioblastos/citologia , Fosforilação OxidativaRESUMO
Proper regulation of cellular lipid storage and oxidation is indispensable for the maintenance of cellular energy homeostasis and health. Mitochondrial function has been shown to be a main determinant of functional lipid storage and oxidation, which is of particular interest for the adipose tissue, as it is the main site of triacylglyceride storage in lipid droplets (LDs). Recent studies have identified a subpopulation of mitochondria attached to LDs, peridroplet mitochondria (PDM) that can be separated from cytoplasmic mitochondria (CM) by centrifugation. PDM have distinct bioenergetics, proteome, cristae organization and dynamics that support LD build-up, however their role in adipose tissue biology remains largely unexplored. Therefore, understanding the molecular basis of LD homeostasis and their relationship to mitochondrial function and attachment in adipocytes is of major importance.
Assuntos
Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Tecido Adiposo/metabolismo , Animais , Sistema Endócrino/metabolismo , Humanos , Gotículas Lipídicas/ultraestrutura , Mitocôndrias/ultraestrutura , Terapia de Alvo MolecularRESUMO
Obesity results from an imbalance in energy homeostasis, whereby excessive energy intake exceeds caloric expenditure. Energy can be dissipated out of an organism by producing heat (thermogenesis), explaining the long-standing interest in exploiting thermogenic processes to counteract obesity. Mitochondrial uncoupling is a process that expends energy by oxidizing nutrients to produce heat, instead of ATP synthesis. Energy can also be dissipated through mechanisms that do not involve mitochondrial uncoupling. Such mechanisms include futile cycles described as metabolic reactions that consume ATP to produce a product from a substrate but then converting the product back into the original substrate, releasing the energy as heat. Energy dissipation driven by cellular ATP demand can be regulated by adjusting the speed and number of futile cycles. Energy consuming futile cycles that are reviewed here are lipolysis/fatty acid re-esterification cycle, creatine/phosphocreatine cycle, and the SERCA-mediated calcium import and export cycle. Their reliance on ATP emphasizes that mitochondrial oxidative function coupled to ATP synthesis, and not just uncoupling, can play a role in thermogenic energy dissipation. Here, we review ATP consuming futile cycles, the evidence for their function in humans, and their potential employment as a strategy to dissipate energy and counteract obesity.
Assuntos
Tecido Adiposo Marrom , Metabolismo Energético , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/metabolismo , Humanos , Obesidade/metabolismo , Ciclização de Substratos , TermogêneseRESUMO
The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane-specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single-particle tracking (SPT), live-cell STED and high-resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.
Assuntos
Membranas Mitocondriais , Proteínas Mitocondriais , Células HeLa , Humanos , Mitocôndrias , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP-consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.
Assuntos
Adipócitos Marrons , Ácido Pirúvico , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Lipídeos , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Termogênese , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Assuntos
Transformação Celular Neoplásica , Metilação de DNA , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Inativação Gênica , Genes Supressores de Tumor , Glicina/metabolismo , Glicólise , Humanos , Camundongos , Ductos Pancreáticos/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Retroelementos/genética , S-Adenosilmetionina/metabolismo , Serina/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Transaminases/metabolismoRESUMO
Assessing mitochondrial function in cell-based systems is a central component of metabolism research. However, the selection of an initial measurement technique may be complicated given the range of parameters that can be studied and the need to define the mitochondrial (dys)function of interest. This methods-focused review compares and contrasts the use of mitochondrial membrane potential measurements, plate-based respirometry, and metabolomics and stable isotope tracing. We demonstrate how measurements of 1) cellular substrate preference, 2) respiratory chain activity, 3) cell activation, and 4) mitochondrial biogenesis are enriched by integrating information from multiple methods. This manuscript is meant to serve as a perspective to help choose which technique might be an appropriate initial method to answer a given question, as well as provide a broad "roadmap" for designing follow-up assays to enrich datasets or resolve ambiguous results.
Assuntos
Bioensaio , Metabolismo Energético , Potencial da Membrana Mitocondrial , Metabolômica , Mitocôndrias/metabolismo , Biogênese de Organelas , Animais , Biomarcadores/metabolismo , Linhagem Celular , Respiração Celular , Humanos , Marcação por Isótopo , Consumo de OxigênioRESUMO
Coenzyme Q (Q n ) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.
Assuntos
Deleção de Genes , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquinona/análogos & derivados , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biossíntese , Ubiquinona/deficiência , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a 'basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.