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1.
Cell ; 175(1): 117-132.e21, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30197082

RESUMO

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.


Assuntos
Proteínas de Transporte/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Metabolismo dos Carboidratos/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Glicólise/fisiologia , Humanos , Ácido Hialurônico/fisiologia , Hialuronoglucosaminidase/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais , Tristetraprolina/metabolismo , Tristetraprolina/fisiologia
2.
Cell ; 168(1-2): 210-223.e11, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28086092

RESUMO

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development.


Assuntos
Ciclo do Ácido Cítrico , Genoma , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Epigênese Genética , Glicosilação , Histonas/metabolismo , Cetona Oxirredutases/metabolismo , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo
3.
Cell ; 151(2): 333-343, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23063123

RESUMO

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.


Assuntos
DNA Mitocondrial/genética , Camundongos/genética , Animais , Comportamento Animal , Cognição , Feminino , Padrões de Herança , Masculino , Camundongos/fisiologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Especificidade da Espécie
4.
Proc Natl Acad Sci U S A ; 109(49): 20065-70, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-23129651

RESUMO

An animal model of Leber hereditary optic neuropathy (LHON) was produced by introducing the human optic atrophy mtDNA ND6 P25L mutation into the mouse. Mice with this mutation exhibited reduction in retinal function by elecroretinogram (ERG), age-related decline in central smaller caliber optic nerve fibers with sparing of larger peripheral fibers, neuronal accumulation of abnormal mitochondria, axonal swelling, and demyelination. Mitochondrial analysis revealed partial complex I and respiration defects and increased reactive oxygen species (ROS) production, whereas synaptosome analysis revealed decreased complex I activity and increased ROS but no diminution of ATP production. Thus, LHON pathophysiology may result from oxidative stress.


Assuntos
DNA Mitocondrial/genética , Modelos Animais de Doenças , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/fisiopatologia , Estresse Oxidativo/fisiologia , Retina/patologia , Trifosfato de Adenosina/metabolismo , Fatores Etários , Animais , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/patologia , Eletrorretinografia , Humanos , Immunoblotting , Camundongos , Mutação de Sentido Incorreto/genética , Atrofia Óptica Hereditária de Leber/complicações , Nervo Óptico/patologia , Espécies Reativas de Oxigênio/metabolismo , Sinaptossomos/metabolismo
5.
Proc Natl Acad Sci U S A ; 109(19): 7391-6, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22517755

RESUMO

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a "complex" disease.


Assuntos
Altitude , DNA Mitocondrial/genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Polimorfismo Genético , Alelos , Substituição de Aminoácidos , Povo Asiático/genética , Linhagem Celular Tumoral , DNA Mitocondrial/química , Frequência do Gene , Predisposição Genética para Doença/genética , Haplótipos , Humanos , Dados de Sequência Molecular , NADH Desidrogenase/metabolismo , Atrofia Óptica Hereditária de Leber/etnologia , Atrofia Óptica Hereditária de Leber/metabolismo , Consumo de Oxigênio , Análise de Sequência de DNA , Tibet
6.
Dev Cell ; 56(16): 2329-2347.e6, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34428399

RESUMO

Mammalian preimplantation embryos follow a stereotypic pattern of development from zygotes to blastocysts. Here, we use labeled nutrient isotopologue analysis of small numbers of embryos to track downstream metabolites. Combined with transcriptomic analysis, we assess the capacity of the embryo to reprogram its metabolism through development. Early embryonic metabolism is rigid in its nutrient requirements, sensitive to reductive stress and has a marked disequilibrium between two halves of the TCA cycle. Later, loss of maternal LDHB and transcription of zygotic products favors increased activity of bioenergetic shuttles, fatty-acid oxidation and equilibration of the TCA cycle. As metabolic plasticity peaks, blastocysts can develop without external nutrients. Normal developmental metabolism of the early embryo is distinct from cancer metabolism. However, similarities emerge upon reductive stress. Increased metabolic plasticity with maturation is due to changes in redox control mechanisms and to transcriptional reprogramming of later-stage embryos during homeostasis or upon adaptation to environmental changes.


Assuntos
Adaptação Fisiológica , Blastocisto/metabolismo , Metaboloma , Animais , Células Cultivadas , Ciclo do Ácido Cítrico , Glucose/metabolismo , Glutamina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Oxirredução , Transcriptoma
7.
Elife ; 102021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34713801

RESUMO

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Hematopoese , Hemócitos/metabolismo , Hemolinfa/metabolismo , Hormônios Juvenis/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Hormônios Juvenis/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Masculino
8.
Dev Cell ; 53(1): 9-26.e4, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32197068

RESUMO

The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation. Furthermore, glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1. In addition, glucose-dependent nucleotide synthesis by the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of Tfap2c. YAP1, TEAD4, and TFAP2C interact to form a complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, and this process of developmental metabolism specifically controls TE cell fate.


Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Proteínas de Homeodomínio/metabolismo , Animais , Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Fatores de Transcrição/metabolismo
9.
Biochemistry ; 48(9): 2053-62, 2009 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-19220002

RESUMO

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron-sulfur clusters. The mechanisms of NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. Studies of ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as ubiquinone-1). Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A). Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation. Moreover, semiquinones produced at the flavin site initiate redox cycling reactions with molecular oxygen, producing superoxide radicals and hydrogen peroxide. The ubiquinone reactant is regenerated, so the NADH:Q reaction becomes superstoichiometric. Idebenone, an artificial ubiquinone showing promise in the treatment of Friedreich's Ataxia, reacts at the flavin site. The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed. Finally, the possibility that the flavin site in complex I catalyzes redox cycling reactions with a wide range of compounds, some of which are important in pharmacology and toxicology, is discussed.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Flavinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/metabolismo , Algoritmos , Animais , Bovinos , Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Cinética , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , NAD/metabolismo , Oxirredução , Oxigênio/metabolismo , Rotenona/farmacologia , Ubiquinona/química , Desacopladores/farmacologia , Água/química
10.
J Biol Chem ; 282(20): 14708-18, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17369262

RESUMO

MitoQ(10) is a ubiquinone that accumulates within mitochondria driven by a conjugated lipophilic triphenylphosphonium cation (TPP(+)). Once there, MitoQ(10) is reduced to its active ubiquinol form, which has been used to prevent mitochondrial oxidative damage and to infer the involvement of reactive oxygen species in signaling pathways. Here we show MitoQ(10) is effectively reduced by complex II, but is a poor substrate for complex I, complex III, and electron-transferring flavoprotein (ETF):quinone oxidoreductase (ETF-QOR). This differential reactivity could be explained if the bulky TPP(+) moiety sterically hindered access of the ubiquinone group to enzyme active sites with a long, narrow access channel. Using a combination of molecular modeling and an uncharged analog of MitoQ(10) with similar sterics (tritylQ(10)), we infer that the interaction of MitoQ(10) with complex I and ETF-QOR, but not complex III, is inhibited by its bulky TPP(+) moiety. To explain its lack of reactivity with complex III we show that the TPP(+) moiety of MitoQ(10) is ineffective at quenching pyrene fluorophors deeply buried within phospholipid bilayers and thus is positioned near the membrane surface. This superficial position of the TPP(+) moiety, as well as the low solubility of MitoQ(10) in non-polar organic solvents, suggests that the concentration of the entire MitoQ(10) molecule in the membrane core is very limited. As overlaying MitoQ(10) onto the structure of complex III indicates that MitoQ(10) cannot react with complex III without its TPP(+) moiety entering the low dielectric of the membrane core, we conclude that the TPP(+) moiety does anchor the tethered ubiquinol group out of reach of the active site(s) of complex III, thus explaining its slow oxidation. In contrast the ubiquinone moiety of MitoQ(10) is able to quench fluorophors deep within the membrane core, indicating a high concentration of the ubiquinone moiety within the membrane and explaining its good anti-oxidant efficacy. These findings will facilitate the rational design of future mitochondria-targeted molecules.


Assuntos
Antioxidantes/química , Complexo I de Transporte de Elétrons/química , Bicamadas Lipídicas/química , Mitocôndrias Cardíacas/enzimologia , Compostos Organofosforados/química , Fosfolipídeos/química , Ubiquinona/análogos & derivados , Animais , Antioxidantes/farmacologia , Bovinos , Complexo I de Transporte de Elétrons/metabolismo , Bicamadas Lipídicas/metabolismo , Compostos Organofosforados/farmacologia , Oxirredução , Fosfolipídeos/metabolismo , Ubiquinona/química , Ubiquinona/farmacologia
11.
J Biol Chem ; 281(46): 34803-9, 2006 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-16980308

RESUMO

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn2+ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn2+ can be used to probe the mechanism of complex I. Zn2+ inhibits complex I more strongly than Mg2+, Ca2+, Ba2+, and Mn2+ to Cu2+ or Cd2+. It does not inhibit NADH oxidation or intramolecular electron transfer, so it probably inhibits either proton transfer to bound quinone or proton translocation. Thus, zinc represents a new class of complex I inhibitor clearly distinct from the many ubiquinone site inhibitors. No evidence for increased superoxide production by zinc-inhibited complex I was detected. Zinc binding to complex I is mechanistically complicated. During catalysis, zinc binds slowly and progressively, but it binds rapidly and tightly to the resting state(s) of the enzyme. Reactivation of the inhibited enzyme upon the addition of EDTA is slow, and inhibition is only partially reversible. The IC50 value for the Zn2+ inhibition of complex I is high (10-50 microm, depending on the enzyme state); therefore, complex I is unlikely to be a major site for zinc inhibition of the electron transport chain. However, the slow response of complex I to a change in Zn2+ concentration may enhance any physiological consequences.


Assuntos
Complexo I de Transporte de Elétrons/antagonistas & inibidores , Mitocôndrias Cardíacas/enzimologia , Zinco/farmacologia , Animais , Cálcio , Bovinos , Complexo I de Transporte de Elétrons/metabolismo
12.
Biochemistry ; 45(1): 241-8, 2006 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-16388600

RESUMO

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, energy transducing, membrane-bound enzyme. It contains 46 different subunits and nine redox cofactors: a noncovalently bound flavin mononucleotide and eight iron-sulfur clusters. The mechanism of complex I is not known. Mechanistic studies using the bovine enzyme, a model for human complex I, have been precluded by the difficulty of preparing complex I which is pure, monodisperse, and fully catalytically active. Here, we describe and characterize a preparation of bovine complex I which fulfills all of these criteria. The catalytic activity is strongly dependent on the phospholipid content of the preparation, and three classes of phospholipid interactions with complex I have been identified. First, complex I contains tightly bound cardiolipin. Cardiolipin may be required for the structural integrity of the complex or play a functional role. Second, the catalytic activity is determined by the amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are bound to the complex. They are more weakly bound than cardiolipin, exchange with PC and PE in solution, and can substitute for one another. However, their nontransitory loss leads to irreversible functional impairment. Third, phospholipids are also required in the assay buffer for the purified enzyme to exhibit its full activity. It is likely that they are required for solubilization and presentation of the hydrophobic ubiquinone substrate.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , NAD/metabolismo , Oxirredutases/metabolismo , Fosfolipídeos/metabolismo , Ubiquinona/metabolismo , Animais , Cardiolipinas/metabolismo , Catálise , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Solubilidade
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