RESUMO
Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.
Assuntos
Mitocôndrias , Neuroblastoma , Apoptose , Ceramidas , Resistência a Múltiplos Medicamentos , Humanos , Membranas Mitocondriais , Neuroblastoma/tratamento farmacológicoRESUMO
SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.
Assuntos
Deficiência de Citocromo-c Oxidase , Doença de Leigh , Animais , Adulto , Humanos , Doença de Leigh/tratamento farmacológico , Doença de Leigh/genética , Doença de Leigh/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Acetilcisteína , Cisteamina/farmacologia , Azidas/metabolismo , Morte Encefálica , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutationa/metabolismo , LactatosRESUMO
Mitochondrial respiratory chain disorders are empirically managed with variable antioxidant, cofactor and vitamin 'cocktails'. However, clinical trial validated and approved compounds, or doses, do not exist for any single or combinatorial mitochondrial disease therapy. Here, we sought to pre-clinically evaluate whether rationally designed mitochondrial medicine combinatorial regimens might synergistically improve survival, health and physiology in translational animal models of respiratory chain complex I disease. Having previously demonstrated that gas-1(fc21) complex I subunit ndufs2-/-C. elegans have short lifespan that can be significantly rescued with 17 different metabolic modifiers, signaling modifiers or antioxidants, here we evaluated 11 random combinations of these three treatment classes on gas-1(fc21) lifespan. Synergistic rescue occurred only with glucose, nicotinic acid and N-acetylcysteine (Glu + NA + NAC), yielding improved mitochondrial membrane potential that reflects integrated respiratory chain function, without exacerbating oxidative stress, and while reducing mitochondrial stress (UPRmt) and improving intermediary metabolic disruptions at the levels of the transcriptome, steady-state metabolites and intermediary metabolic flux. Equimolar Glu + NA + NAC dosing in a zebrafish vertebrate model of rotenone-based complex I inhibition synergistically rescued larval activity, brain death, lactate, ATP and glutathione levels. Overall, these data provide objective preclinical evidence in two evolutionary-divergent animal models of mitochondrial complex I disease to demonstrate that combinatorial Glu + NA + NAC therapy significantly improved animal resiliency, even in the face of stressors that cause severe metabolic deficiency, thereby preventing acute neurologic and biochemical decompensation. Clinical trials are warranted to evaluate the efficacy of this lead combinatorial therapy regimen to improve resiliency and health outcomes in human subjects with mitochondrial disease.
Assuntos
Acetilcisteína/farmacologia , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Glucose/farmacologia , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/prevenção & controle , Niacina/farmacologia , Animais , Caenorhabditis elegans , Sinergismo Farmacológico , Complexo I de Transporte de Elétrons/genética , Sequestradores de Radicais Livres/farmacologia , Humanos , Longevidade/efeitos dos fármacos , Longevidade/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Mutação , Estresse Oxidativo/efeitos dos fármacos , Peixe-ZebraRESUMO
Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.
Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Epigenoma , Acetilcoenzima A/metabolismo , Linhagem Celular , Histonas/metabolismo , Humanos , Metaboloma , Mitocôndrias/metabolismo , NAD/metabolismo , Transcrição GênicaRESUMO
Cysteamine bitartrate is a US Food and Drug Administration-approved therapy for nephropathic cystinosis also postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionarily distinct species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in Caenorhabditis elegans gas-1(fc21) RC complex I (NDUFS2-/-) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 µm concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) Danio rerio vertebrate zebrafish disease models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways was greater in RC disease human cells than in C. elegans, with similar rescue in both models of downregulated ribosomal and proteasomal pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.
Assuntos
Antioxidantes/farmacologia , Proteínas de Caenorhabditis elegans/genética , Cisteamina/farmacologia , Doenças Mitocondriais/tratamento farmacológico , NADH Desidrogenase/genética , Animais , Morte Encefálica/metabolismo , Morte Encefálica/patologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Proteínas F-Box/genética , Fertilidade/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Glutationa/genética , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Estresse Oxidativo/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genéticaRESUMO
Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.
Assuntos
Gluconobacter oxydans/enzimologia , NADH Desidrogenase/metabolismo , NADP/metabolismo , NAD/metabolismo , Gluconobacter oxydans/genética , Gluconobacter oxydans/metabolismo , Homeostase , Niacinamida/metabolismo , Oxirredução , Periplasma/metabolismoRESUMO
Separation of the rare-earth (RE) elements (Sc, Y, La-Lu) is challenging because of their similar chemical properties, but is necessary for their applications in renewable energy and electronic device technologies. The development of separation processes driven by kinetic factors represents a new area for this field. Herein, we disclose a novel method of separating select rare earths by reacting RE cyclopentadienides with the triradical species tris(2-tert-butylnitroxyl)benzylamine (1). The key proligand 1 was characterized using a variety of techniques including X-ray crystallography, magnetometry, and EPR spectroscopy. When applied to an equimolar mixture of La:Y cyclopentadienide complexes, different rates of chelation of these organometallic precursors by 1 were observed, affording a separation factor of 26 under the reported conditions.
RESUMO
Methyl benzoate, N,N-dimethylbenzamide, and benzophenone were reduced by UIII [N(SiMe3 )2 ]3 resulting in uranium(IV) products. Reduction of benzophenone lead to UIV [OCâ Ph2 )][N(SiMe3 )2 ]3 , (1.1) which forms the dinuclear complex, [N(SiMe3 )2 ]3 UIV (OCPhPh-CPh2 O)UIV [N(SiMe3 )2 ]3 (1.2), through coupling of the ketyl radical species upon crystallization. Reaction of N,N-dimethylbenzamide with UIII [N(SiMe3 )2 ]3 resulted in UIV [OCâ (Ph)(NMe2 )][N(SiMe3 )2 ]3 (2), a uranium(IV) compound and the first example of a charge-separated amide radical. In the case of methyl benzoate, the reduction resulted in UIV (OMe)[N(SiMe3 )2 ]3 (3) and benzaldehyde as the reduced organic fragment. Compound 2 showed the ability to act as a uranium(III) synthon in its reactivity with trimethylsilyl azide, a reaction that yielded UV (=NSiMe3 )[N(SiMe3 )2 ]3 . Additionally, 2 was reduced with potassium graphite resulting in [U(µ-O)[O=C(NMe2 )(Ph)][N(SiMe3 )2 ]2 ]2 (4), a dinuclear uranium compound bridged by oxo ligands. Reduction of 2 in the presence of 15-crown-5 afforded isolation of the mono-oxo compound, [(15-crown-5)2 K][UO[N(SiMe3 )2 ]3 ] (5). The results expand the reduction capabilities of UIII complexes and demonstrate a strategy for isolating novel metal-stabilized radicals.
RESUMO
BACKGROUND: Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. METHODS: MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. RESULTS: LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl-. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. CONCLUSIONS: We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.
Assuntos
Butileno Glicóis/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Leucócitos/enzimologia , Macrófagos/enzimologia , Neutrófilos/enzimologia , Peroxidase/antagonistas & inibidores , Animais , Catálise , Células Cultivadas , Humanos , Leucócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , OxirreduçãoRESUMO
Respiratory complex I transfers electrons from NADH to quinone, utilizing the reaction energy to translocate protons across the membrane. It is a key enzyme of the respiratory chain of many prokaryotic and most eukaryotic organisms. The reversible NADH oxidation reaction is facilitated in complex I by non-covalently bound flavin mononucleotide (FMN). Here we report that the catalytic activity of E. coli complex I with artificial electron acceptors potassium ferricyanide (FeCy) and hexaamineruthenium (HAR) is significantly inhibited in the enzyme pre-reduced by NADH. Further, we demonstrate that the inhibition is caused by reversible dissociation of FMN. The binding constant (Kd) for FMN increases from the femto- or picomolar range in oxidized complex I to the nanomolar range in the NADH reduced enzyme, with an FMN dissociation time constant of ~5s. The oxidation state of complex I, rather than that of FMN, proved critical to the dissociation. Such dissociation is not observed with the T. thermophilus enzyme and our analysis suggests that the difference may be due to the unusually high redox potential of Fe-S cluster N1a in E. coli. It is possible that the enzyme attenuates ROS production in vivo by releasing FMN under highly reducing conditions.
Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas de Escherichia coli/química , Mononucleotídeo de Flavina/química , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Oxirredução , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
UNLABELLED: Merkel cell polyomavirus (MCPyV) plays an important role in Merkel cell carcinoma (MCC). MCPyV small T (sT) antigen has emerged as the key oncogenic driver in MCC carcinogenesis. It has also been shown to promote MCPyV LT-mediated replication by stabilizing LT. The importance of MCPyV sT led us to investigate sT functions and to identify potential ways to target this protein. We discovered that MCPyV sT purified from bacteria contains iron-sulfur (Fe/S) clusters. Electron paramagnetic resonance analysis showed that MCPyV sT coordinates a [2Fe-2S] and a [4Fe-4S] cluster. We also observed phenotypic conservation of Fe/S coordination in the sTs of other polyomaviruses. Since Fe/S clusters are critical cofactors in many nucleic acid processing enzymes involved in DNA unwinding and polymerization, our results suggested the hypothesis that MCPyV sT might be directly involved in viral replication. Indeed, we demonstrated that MCPyV sT enhances LT-mediated replication in a manner that is independent of its previously reported ability to stabilize LT. MCPyV sT translocates to nuclear foci containing actively replicating viral DNA, supporting a direct role for sT in promoting viral replication. Mutations of Fe/S cluster-coordinating cysteines in MCPyV sT abolish its ability to stimulate viral replication. Moreover, treatment with cidofovir, a potent antiviral agent, robustly inhibits the sT-mediated enhancement of MCPyV replication but has little effect on the basal viral replication driven by LT alone. This finding further indicates that MCPyV sT plays a direct role in stimulating viral DNA replication and introduces cidofovir as a possible drug for controlling MCPyV infection. IMPORTANCE: MCPyV is associated with a highly aggressive form of skin cancer in humans. Epidemiological surveys for MCPyV seropositivity and sequencing analyses of healthy human skin suggest that MCPyV may represent a common component of the human skin microbial flora. However, much of the biology of the virus and its oncogenic ability remain to be investigated. In this report, we identify MCPyV sT as a novel Fe/S cluster protein and show that conserved cysteine clusters are important for sT's ability to enhance viral replication. Moreover, we show that sT sensitizes MCPyV replication to cidofovir inhibition. The discovery of Fe/S clusters in MCPyV sT opens new avenues to the study of the structure and functionality of this protein. Moreover, this study supports the notion that sT is a potential drug target for dampening MCPyV infection.
Assuntos
Antígenos Virais de Tumores/metabolismo , Replicação do DNA , Proteínas Ferro-Enxofre/metabolismo , Poliomavírus das Células de Merkel/fisiologia , Replicação Viral , Antígenos Virais de Tumores/química , Antígenos Virais de Tumores/isolamento & purificação , Antivirais/metabolismo , Linhagem Celular , Núcleo Celular/química , Cidofovir , Citosina/análogos & derivados , Citosina/metabolismo , Análise Mutacional de DNA , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/isolamento & purificação , Poliomavírus das Células de Merkel/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Organofosfonatos/metabolismo , Transporte ProteicoRESUMO
Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = â¼1 µm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.
Assuntos
Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Proteínas de Membrana/genética , Proteínas Mitocondriais/metabolismo , Fator de Indução de Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Clonagem Molecular , Complexo I de Transporte de Elétrons/genética , Escherichia coli/genética , Expressão Gênica , Biblioteca Gênica , Teste de Complementação Genética , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Cinética , Proteínas de Membrana/deficiência , Membranas Mitocondriais , Proteínas Mitocondriais/genética , Mutação , NAD/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a â¼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.
Assuntos
Citocinas/metabolismo , Músculo Esquelético/metabolismo , NAD/biossíntese , Nicotinamida Fosforribosiltransferase/metabolismo , Oxigênio/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cromatografia Líquida de Alta Pressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Musculares/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Oxirredução , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Complex I (NADH:quinone oxidoreductase) is central to cellular aerobic energy metabolism, and its deficiency is involved in many human mitochondrial diseases. Complex I translocates protons across the membrane using electron transfer energy. Semiquinone (SQ) intermediates appearing during catalysis are suggested to be key for the coupling mechanism in complex I. However, the existence of SQ has remained controversial due to the extreme difficulty in detecting unstable and low intensity SQ signals. Here, for the first time with Escherichia coli complex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQNf) with P1/2 (half-saturation power level)>50mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQNs) with P1/2=2-3mW and a 10G linewidth; and very slow-relaxing SQ (SQNvs) with P1/2= ~0.1mW and a 7.5G linewidth. The SQNf signals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potent E. coli complex I inhibitor. The pH dependency of the SQNf signals correlated with the proton-pumping activities of complex I. The SQNs signals were insensitive to gramicidin D, but sensitive to squamotacin. The SQNvs signals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQNf is neutral, while SQNs and SQNvs are anion radicals. The SQNs signals were lost in the ΔNuoL mutant missing transporter module subunits NuoL and NuoM. The roles and relationships of the SQ intermediates in the coupling mechanism are discussed.
Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , NADH Desidrogenase/química , Prótons , Ubiquinona/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Antibacterianos/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gramicidina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Oxirredução , Proteolipídeos , Ubiquinona/metabolismoRESUMO
Serotonin (5-HT) is a well-known biogenic amine which regulates mood, sleep, and is involved in muscle contraction and blood coagulation. Based on an analogy to norepinephrine, a ß-hydroxylated derivative of dopamine which has diverse physiological functions, beta-hydroxy-serotonin (ß-OH-5-HT) originally encouraged interest as a potential pharmacological agent. Four decades ago, its organic synthesis was attempted. However, due to difficulties with the synthesis and the compound's instability, rigorous identification and characterization of ß-OH-5-HT proved evasive. Here, we successfully synthesized ß-OH-5-HT from 5-HT using a Pseudomonas enzyme, tryptophan side chain oxidase type I (TSOI), and we determined the structure by 2D-NMR and characterized ß-OH-5-HT in detail. The CD spectra showed no optical activity, suggesting a racemic mixture. To separate DL-ß-OH-5-HT, we synthesized L-Ala-5-HT and derivatized it into erythro- and threo-L-Ala-ß-OH-5-HT with TSOI. Interestingly, both isolated fractions returned to a diastereoisomeric mixture within two hours at pH 5.0. Later, we found that, under acidic conditions, ß-OH-5-HT readily reacted with nucleophiles like alcohols or thiols, yielding a variety of DL-ß-substituted-5-HT. The unusual properties of ß-OH-5-HT might be attributed to the unique nature of a ß-hydroxyl group adjacent to an indole ring and amino group. The mechanism for the rapid racemization of ß-OH-5-HT is discussed.
Assuntos
Serotonina/análogos & derivados , Hidroxilação , Isomerismo , Estrutura Molecular , Serotonina/síntese químicaRESUMO
NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 µmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Quinonas/química , Análise por Conglomerados , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Flavinas/química , Histidina/química , Concentração de Íons de Hidrogênio , Proteínas Ferro-Enxofre/química , Mutação , Oxirredução , Bombas de Próton/químicaRESUMO
Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ(Nf)) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ(Ns)) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ(Nvs)) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ(Ns) signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.
Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , NADH Desidrogenase/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , Antibacterianos/química , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gramicidina/química , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
Systematic ligand variation in a structurally conserved framework of pentavalent uranium complexes of the formulas U(V)X2[N(SiMe3)2]3 (X = F, Cl, Br, N3, NCS, 2-naphthoxide) and U(V)OX[N(SiMe3)2]3(-) (X = -CCPh, -CN) allowed an investigation into the role of the inverse trans influence in pentavalent uranium complexes. The -CCPh and -CN derivatives were only stable in the presence of the trans-UâO multiple bond, implicating the inverse trans influence in stabilizing these complexes. Spectroscopic, structural, and density functional theory calculated electronic structural data are explored. Near-IR data of all complexes is presented, displaying vibronic coupling of 5f(1) electronic transitions along the primary axis. Electrochemical characterization allowed assessment of the relative donating ability of the various axial ligands in this framework. Electron paramagnetic resonance data presented display axial spectra, with hyperfine coupling along the primary axis.
Assuntos
Compostos de Urânio/síntese química , Eletroquímica , Elétrons , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Compostos de Urânio/química , Difração de Raios XRESUMO
N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.
RESUMO
In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus generates a 24 h rhythm of sleep and arousal. While neuronal spiking activity in the SCN provides a functional circadian oscillator that propagates throughout the brain, the ultradian sleep-wake state is regulated by the basal forebrain/preoptic area (BF/POA). How this SCN circadian oscillation is integrated into the shorter sleep-wake cycles remains unclear. We examined the temporal patterns of neuronal activity in these key brain regions in freely behaving rats. Neuronal activity in various brain regions presented diurnal rhythmicity and/or sleep-wake state dependence. We identified a diurnal rhythm in the BF/POA that was selectively degraded when diurnal arousal patterns were disrupted by acute brain serotonin depletion despite robust circadian spiking activity in the SCN. Local blockade of serotonergic transmission in the BF/POA was sufficient to disrupt the diurnal sleep-wake rhythm of mice. These results suggest that the serotonergic system enables the BF/POA to couple the SCN circadian signal to ultradian sleep-wake cycles, thereby providing a potential link between circadian rhythms and psychiatric disorders.