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1.
Science ; 371(6524): 52-57, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33384370

RESUMO

Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.


Assuntos
Diferenciação Celular , Hipóxia/patologia , Células Neuroendócrinas/fisiologia , Oxigênio/fisiologia , Células-Tronco/fisiologia , Traqueia/citologia , Anaerobiose , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Contagem de Células , Deleção de Genes , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Mutantes , Células Neuroendócrinas/citologia , Prolil Hidroxilases/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transativadores/genética
2.
J Clin Invest ; 131(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33463549

RESUMO

Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, ß-hydroxy acylcarnitines, and ß-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

3.
Nucleic Acids Res ; 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33174596

RESUMO

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

4.
N Engl J Med ; 383(16): 1556-1563, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32786181

RESUMO

Many mitochondrial diseases are caused by mutations in mitochondrial DNA (mtDNA). Patients' cells contain a mixture of mutant and nonmutant mtDNA (a phenomenon called heteroplasmy). The proportion of mutant mtDNA varies across patients and among tissues within a patient. We simultaneously assayed single-cell heteroplasmy and cell state in thousands of blood cells obtained from three unrelated patients who had A3243G-associated mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes. We observed a broad range of heteroplasmy across all cell types but also found markedly reduced heteroplasmy in T cells, a finding consistent with purifying selection within this lineage. We observed this pattern in six additional patients who had heteroplasmic A3243G without strokelike episodes. (Funded by the Marriott Foundation and others.).


Assuntos
DNA Mitocondrial/genética , Mutação , Polimorfismo Genético , Linfócitos T , Adulto , Genoma Mitocondrial , Humanos , Leucócitos Mononucleares , Síndrome MELAS/genética , Masculino , Pessoa de Meia-Idade
5.
Elife ; 92020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32762847

RESUMO

Mitochondrial Ca2+ uptake is mediated by an inner mitochondrial membrane protein called the mitochondrial calcium uniporter. In humans, the uniporter functions as a holocomplex consisting of MCU, EMRE, MICU1 and MICU2, among which MCU and EMRE form a subcomplex and function as the conductive channel while MICU1 and MICU2 are EF-hand proteins that regulate the channel activity in a Ca2+-dependent manner. Here, we present the EM structures of the human mitochondrial calcium uniporter holocomplex (uniplex) in the presence and absence of Ca2+, revealing distinct Ca2+ dependent assembly of the uniplex. Our structural observations suggest that Ca2+ changes the dimerization interaction between MICU1 and MICU2, which in turn determines how the MICU1-MICU2 subcomplex interacts with the MCU-EMRE channel and, consequently, changes the distribution of the uniplex assemblies between the blocked and unblocked states.

6.
Life Sci Alliance ; 3(10)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32769116

RESUMO

The mitochondrial calcium uniporter (MCU) is a calcium-activated calcium channel critical for signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan protein essential MCU regulator (EMRE) for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of human MCU's dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent Dictyostelium discoideum MCU and Homo sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in D. discoideum MCU that can be transplanted to HsMCU to render it EMRE independent. We call this region in human MCU the EMRE dependence domain (EDD). Crosslinking experiments show that EMRE directly interacts with HsMCU at its transmembrane domains as well as the EDD. Our results suggest that EMRE stabilizes the EDD of MCU, permitting both channel opening and calcium conductance, consistent with recently published structures of MCU-EMRE.

7.
Nature ; 583(7817): 631-637, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641830

RESUMO

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.


Assuntos
Toxinas Bacterianas/metabolismo , Citidina Desaminase/metabolismo , DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Mitocôndrias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sequência de Bases , Burkholderia cenocepacia/enzimologia , Burkholderia cenocepacia/genética , Respiração Celular/genética , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Genoma Mitocondrial/genética , Células HEK293 , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/terapia , Mutação , Fosforilação Oxidativa , Engenharia de Proteínas , RNA Guia/genética , Especificidade por Substrato , Sistemas de Secreção Tipo VI/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
Nature ; 583(7814): 122-126, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32461692

RESUMO

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.


Assuntos
Fígado/metabolismo , NAD/metabolismo , Estresse Fisiológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citosol/metabolismo , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/sangue , Variação Genética , Teste de Tolerância a Glucose , Humanos , Resistência à Insulina , Lactobacillus brevis/enzimologia , Lactobacillus brevis/genética , Masculino , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Triglicerídeos/sangue
10.
Elife ; 92020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32463360

RESUMO

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.

12.
Cell ; 181(3): 716-727.e11, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32259488

RESUMO

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

13.
Nat Biotechnol ; 38(3): 309-313, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932725

RESUMO

An elevated intracellular NADH:NAD+ ratio, or 'reductive stress', has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD+ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD+ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD+ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.


Assuntos
Bactérias/enzimologia , Catalase/metabolismo , Ácido Láctico/sangue , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Bactérias/metabolismo , Células HeLa , Humanos , Células K562 , NAD/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
14.
Science ; 366(6465): 589-593, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672889

RESUMO

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5'-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.


Assuntos
Coenzima A/metabolismo , Metilmalonil-CoA Mutase/antagonistas & inibidores , Metilmalonil-CoA Mutase/metabolismo , Mycobacterium tuberculosis/enzimologia , Succinatos/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Desoxiadenosinas , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligação de Hidrogênio , Macrófagos/metabolismo , Metilmalonil-CoA Mutase/química , Modelos Moleculares , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Propionatos/metabolismo , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Succinatos/farmacologia , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia
15.
Cell ; 179(5): 1222-1238.e17, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31730859

RESUMO

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.


Assuntos
Genes Modificadores , Mitocôndrias/genética , Mitocôndrias/patologia , Autoantígenos/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Epistasia Genética/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Genoma , Glutationa Peroxidase/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Células K562 , Mitocôndrias/efeitos dos fármacos , Oligomicinas/toxicidade , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Espécies Reativas de Oxigênio/metabolismo , Ribonucleoproteínas/metabolismo
16.
Cancer Cell ; 36(4): 369-384.e13, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31543463

RESUMO

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Evolução Clonal/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cell Metab ; 30(4): 824-832.e3, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31402314

RESUMO

Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease. Rather, we observe an age-dependent decline in whole-body oxygen consumption. These mice exhibit brain tissue hyperoxia, which is normalized by hypoxic breathing. Alternative experimental strategies to reduce oxygen delivery, including breathing carbon monoxide (600 ppm in air) or severe anemia, can reverse neurological disease. Therefore, unused oxygen is the most likely culprit in the pathology of this disease. While pharmacologic activation of the hypoxia response is unlikely to alleviate disease in vivo, interventions that safely normalize brain tissue hyperoxia may hold therapeutic potential.


Assuntos
Encéfalo/metabolismo , Monóxido de Carbono/uso terapêutico , Hiperóxia/terapia , Doença de Leigh/terapia , Oxigênio/metabolismo , Anemia/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Hiperóxia/metabolismo , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Doença de Leigh/metabolismo , Camundongos
19.
Cell Metab ; 30(3): 539-555.e11, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31257153

RESUMO

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.


Assuntos
Aminoidrolases/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Ácido Fólico/metabolismo , Herpesvirus Humano 4/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Feminino , Glicólise , Células HEK293 , Humanos , Ativação Linfocitária , Mitocôndrias/metabolismo , NADP/biossíntese , Oxirredução , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina/biossíntese
20.
Cell Rep ; 27(5): 1364-1375.e5, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31042465

RESUMO

The mitochondrial calcium uniporter has been proposed to coordinate the organelle's energetics with calcium signaling. Uniporter current has previously been reported to be extremely high in brown adipose tissue (BAT), yet it remains unknown how the uniporter contributes to BAT physiology. Here, we report the generation and characterization of a mouse model lacking Mcu, the pore forming subunit of the uniporter, specifically in BAT (BAT-Mcu-KO). BAT-Mcu-KO mice lack uniporter-based calcium uptake in BAT mitochondria but exhibit unaffected cold tolerance, diet-induced obesity, and transcriptional response to cold in BAT. Unexpectedly, we found in wild-type animals that cold powerfully activates the ATF4-dependent integrated stress response (ISR) in BAT and upregulates circulating FGF21 and GDF15, raising the hypothesis that the ISR partly underlies the pleiotropic effects of BAT on systemic metabolism. Our study demonstrates that the uniporter is largely dispensable for BAT thermogenesis and demonstrates activation of the ISR in BAT in response to cold.


Assuntos
Tecido Adiposo Marrom/metabolismo , Canais de Cálcio/genética , Resposta ao Choque Frio , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Termogênese , Fator 4 Ativador da Transcrição/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/genética
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