RESUMO
The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is â¼10 µm for mouse and 1 µm for human SAMHD1, for dTTP the corresponding values are 50 and 2 µm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotidases/química , Nucleotidases/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Hidrólise , Cinética , Camundongos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleotidases/genética , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Precursores de Ácido Nucleico/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Replicação do DNA , Inativação Gênica , Humanos , RNA Interferente Pequeno , Proteína 1 com Domínio SAM e Domínio HDRESUMO
In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Replicação do DNA/genética , DNA Mitocondrial/genética , Fibroblastos/enzimologia , Mamíferos/metabolismo , Mitocôndrias/genética , Ribonucleotídeo Redutases/metabolismo , Animais , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Etídio/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Fluorescência , Fluorometria , Histonas/metabolismo , Humanos , Hidroxiureia/metabolismo , Hidroxiureia/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Mutação/genética , Conformação de Ácido Nucleico , Nucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Subunidades Proteicas/metabolismo , Raios UltravioletaRESUMO
Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and DNA repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 and R2. A third protein, p53R2, with the same function as R2, occurs in minute amounts. In quiescent cells, p53R2 replaces the absent R2. In humans, genetic inactivation of p53R2 causes early death with mtDNA depletion, especially in muscle. We found that cycling fibroblasts from a patient with a lethal mutation in p53R2 contained a normal amount of mtDNA and showed normal growth, ribonucleotide reduction, and deoxynucleoside triphosphate (dNTP) pools. However, when made quiescent by prolonged serum starvation the mutant cells strongly down-regulated ribonucleotide reduction, decreased their dCTP and dGTP pools, and virtually abolished the catabolism of dCTP in substrate cycles. mtDNA was not affected. Also, nuclear DNA synthesis and the cell cycle-regulated enzymes R2 and thymidine kinase 1 decreased strongly, but the mutant cell populations retained unexpectedly larger amounts of the two enzymes than the controls. This difference was probably due to their slightly larger fraction of S phase cells and therefore not induced by the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and leads to a decrease of dNTP catabolism by substrate cycles that counterweigh the loss of anabolic activity. We speculate that this compensatory mechanism suffices to maintain mtDNA in fibroblasts but not in muscle cells with a larger content of mtDNA necessary for their high energy requirements.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , DNA Mitocondrial/metabolismo , Desoxirribonucleotídeos/metabolismo , Fibroblastos/enzimologia , Mutação de Sentido Incorreto , Ribonucleotídeo Redutases/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , DNA Mitocondrial/genética , Desoxirribonucleotídeos/genética , Fibroblastos/citologia , Humanos , Oxirredução , Ribonucleotídeo Redutases/genética , Timidina Quinase/genética , Timidina Quinase/metabolismoRESUMO
The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.
Assuntos
Arabinonucleosídeos/metabolismo , Arabinonucleotídeos/metabolismo , Ciclo Celular , Nucleotídeos de Desoxiguanina/metabolismo , Desoxiguanosina/metabolismo , Guanosina Trifosfato/análogos & derivados , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Apoptose/efeitos dos fármacos , Arabinonucleosídeos/farmacologia , Arabinonucleotídeos/biossíntese , Biocatálise , Células CHO , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Citosol/enzimologia , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Nucleotídeos de Desoxiguanina/biossíntese , Desoxiguanosina/farmacologia , Desoxirribonucleotídeos/metabolismo , Fibroblastos/enzimologia , Fase G1/efeitos dos fármacos , Guanosina Trifosfato/biossíntese , Guanosina Trifosfato/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Cinética , Mitocôndrias/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Purina-Núcleosídeo Fosforilase/metabolismo , Fase S/efeitos dos fármacosRESUMO
Deoxyribonucleoside triphosphates (dNTPs) are the precursors used by DNA polymerases for replication and repair of nuclear and mitochondrial DNA in animal cells. Accurate DNA synthesis requires adequate amounts of each dNTP and appropriately balanced dNTP pools. Total cellular pool sizes are in the range of 10-100pmoles of each dNTP/million cells during S phase, with mitochondrial pools representing at most 10% of the total. In quiescent or differentiated cells pools are about 10-fold lower both in the cytosol and mitochondria. Contrary to what may be expected on the basis of the roughly equimolar abundance of the 4 nitrogen bases in DNA, the four dNTPs are present in the pools in different ratios, with pyrimidines often exceeding purines. Individual cell lines may exhibit different pool compositions even if they are derived from the same animal species. It has been known for several decades that imbalance of dNTP pools has mutagenic and cytotoxic effects, and leads to "mutator" phenotypes characterized by increased mutation frequencies. Until 10 years ago this phenomenon was considered to affect exclusively the nuclear genome. With the discovery that thymidine phosphorylase deficiency causes destabilization of mitochondrial DNA and a severe multisystemic syndrome the importance of dNTP pool balance was extended to mitochondria. Following that first discovery, mutations in other genes coding for mitochondrial or cytosolic enzymes of dNTP metabolism have been associated with mitochondrial DNA depletion syndromes. Both excess and deficiency of one dNTP may be detrimental. We study the mechanisms that in mammalian cells keep the dNTP pools in balance, and are particularly interested in the enzymes that, similar to thymidine phosphorylase, contribute to pool regulation by degrading dNTP precursors. The role of some relevant enzymes is illustrated with data obtained by chemical or genetic manipulation of their expression in cultured mammalian cells.
Assuntos
Desoxirribonucleotídeos/metabolismo , Nucleotidases/fisiologia , Timidina Fosforilase/fisiologia , Animais , Ciclo Celular , DNA Mitocondrial/metabolismo , Desoxiguanosina/toxicidade , Dissacarídeos , Eletrólitos , Glutamatos , Glutationa , Histidina , Humanos , Manitol , Mutação , Timidina Quinase/metabolismoRESUMO
Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.
Assuntos
RNA/metabolismo , Ribonucleotídeo Redutases/química , Regulação Alostérica , Catálise , Cristalografia por Raios X , Escherichia coli/enzimologia , Conformação Proteica , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismo , Especificidade por Substrato , Thermotoga maritima/enzimologiaRESUMO
Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.
Assuntos
DNA Polimerase Dirigida por DNA , Desoxirribonucleotídeos/análise , Extratos Celulares/química , Citidina Trifosfato , DNA Polimerase I , Nucleotídeos de Desoxicitosina/análise , Nucleotídeos de Desoxiguanina/análise , Humanos , Ribonucleotídeos , Taq PolimeraseRESUMO
Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.
Assuntos
Citosol/metabolismo , Dano ao DNA , Ribonucleotídeos/metabolismo , Células 3T3 , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Western Blotting , Células COS , Fracionamento Celular , Núcleo Celular/enzimologia , Chlorocebus aethiops , Citosol/enzimologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microscopia Confocal , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleotídeo Redutases/metabolismo , Frações Subcelulares/enzimologia , TransfecçãoRESUMO
Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [(3)H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation.
Assuntos
Núcleo Celular/metabolismo , DNA Mitocondrial/metabolismo , DNA/química , Guanina/química , Mitocôndrias/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Citosol/química , Fibroblastos/metabolismo , Humanos , Cinética , Modelos Biológicos , Nucleotídeos/químicaAssuntos
Bioquímica/história , Prêmio Nobel , Replicação do DNA , História do Século XX , Humanos , Estados UnidosRESUMO
Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Desoxirribonucleotídeos/metabolismo , Ribonucleotídeo Redutases/fisiologia , Western Blotting , Linhagem Celular , DCMP Desaminase/metabolismo , Reparo do DNA , Humanos , Timidilato Sintase/metabolismoRESUMO
Recent publications have suggested that imatinib (Glivec) may be cardiotoxic. We have therefore assessed the largest study on the agent performed in patients with gastrointestinal stromal tumours, randomising a daily dose of 400mg versus 800 mg. 946 Patients were entered, 942 patients received at least one dose of imatinib. The median time on treatment was 24 months. A total of 24,574 exposure months could be analysed. We could not identify an excess of cardiac events in the study population. In 2 patients (0.2%) a possible cardiotoxic effect of imatinib could not fully be excluded. The current analysis of a large randomised prospective study could not confirm previous suggestions of imatinib induced cardiac toxicity.
Assuntos
Antineoplásicos/efeitos adversos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Insuficiência Cardíaca/induzido quimicamente , Piperazinas/efeitos adversos , Pirimidinas/efeitos adversos , Disfunção Ventricular Esquerda/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzamidas , Feminino , Humanos , Mesilato de Imatinib , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Timidina Monofosfato/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos de Timina/metabolismoRESUMO
Ribonucleotide reductases (RNRs) transform RNA building blocks to DNA building blocks by catalyzing the substitution of the 2'OH-group of a ribonucleotide with a hydrogen by a mechanism involving protein radicals. Three classes of RNRs employ different mechanisms for the generation of the protein radical. Recent structural studies of members from each class have led to a deeper understanding of their catalytic mechanism and allosteric regulation by nucleoside triphosphates. The main emphasis of this review is on regulation of RNR at the molecular and cellular level. Conformational transitions induced by nucleotide binding determine the regulation of substrate specificity. An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, together with the allosteric effects, enzyme activity and provides the appropriate amount of deoxynucleotides for DNA replication and repair. In spite of large differences in the amino acid sequences, basic structural features are remarkably similar and suggest a common evolutionary origin for the three classes.
Assuntos
Ribonucleotídeo Redutases , Regulação Alostérica , Animais , Sítios de Ligação , Evolução Molecular , Radicais Livres/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Especificidade por Substrato , Ativação TranscricionalRESUMO
Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine.
Assuntos
DNA Mitocondrial , Gastroenteropatias/patologia , Mitocôndrias/metabolismo , Encefalomiopatias Mitocondriais/patologia , Doenças do Sistema Nervoso/patologia , Nucleotídeos de Timina/metabolismo , Trifosfato de Adenosina/química , Citosol/metabolismo , DNA Mitocondrial/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrólise , Pele/metabolismo , Timidina/metabolismo , Timidina Monofosfato/metabolismoRESUMO
Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.
Assuntos
Proteínas de Algas/metabolismo , Euglena gracilis/enzimologia , Proteínas de Protozoários/metabolismo , Ribonucleotídeo Redutases/metabolismo , Vitamina B 12/metabolismo , Proteínas de Algas/classificação , Proteínas de Algas/genética , Proteínas de Algas/isolamento & purificação , Regulação Alostérica , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/isolamento & purificação , Alinhamento de SequênciaRESUMO
Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.
Assuntos
Desoxirribonucleotídeos/química , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Encefalomiopatias Mitocondriais/patologia , Linhagem Celular , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Meios de Cultura/metabolismo , Citosol/enzimologia , Citosol/metabolismo , DNA Mitocondrial/química , Desoxiuridina/química , Humanos , Encefalomiopatias Mitocondriais/metabolismo , Fosforilação , Timidina/química , Timidina Quinase/metabolismo , Timidina Fosforilase/química , Fatores de TempoRESUMO
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12-dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.