RESUMO
In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Fibroblastos/metabolismo , Mutação/genética , Malformações do Sistema Nervoso/genética , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Dano ao DNA/genética , Replicação do DNA/genética , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genéticaRESUMO
Mitochondria play a central role in cancer development, by contributing to most of the classical hallmarks of cancer, including sustained proliferation, metabolic re-programming, apoptosis resistance, invasion and induction of angiogenesis [1]. In addition, mitochondria affect also the function of anti- and pro-tumoral immune cells in the tumor microenvironment. Mitochondria harbor a plethora of regulated ion channels whose function is related to ion/ metabolite transport and to fine-tuning of mitochondrial membrane potential as well as of reactive oxygen species release. As a consequence, growing evidence link ion channels located both in the outer and inner mitochondrial membranes to several cancer hallmarks. The present review summarizes our recent knowledge about the participation and role of mitochondrial channels leading to acquisition of cancer hallmarks and thus to cancer progression.
Assuntos
Canais Iônicos/metabolismo , Mitocôndrias/patologia , Neoplasias/patologia , Animais , Apoptose , Proliferação de Células , Progressão da Doença , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologiaRESUMO
The efficiency of Nucleotide Excision Repair (NER)process is crucial for maintaining genomic integrity because in many organisms, including humans, it represents the only system able to repair a wide range of DNA damage. The aim of the work was to investigate whether the efficiency of the repair of photoproducts induced by UV-light is affected by the circadian phase at which irradiation occurred. NER activity has been analyzed in human quiescent fibroblasts (in the absence of the cell cycle effect), in which circadian rhythmicity has been synchronized with a pulse of dexamethasone. Our results demonstrate that both DNA damage induction and repair efficiency are strictly dependent on the phase of the circadian rhythm at which the cells are UV-exposed. Furthermore, the differences observed between fibroblasts irradiated at different circadian times (CTs) are abolished when the clock is obliterated. In addition, we observe that chromatin structure is regulated by circadian rhythmicity. Maximal chromatin relaxation occurred at the same CT when photoproduct formation and removal were highest. Our data suggest that the circadian clock regulates both the DNA sensitivity to UV damage and the efficiency of NER by controlling chromatin condensation mainly through histone acetylation.
Assuntos
Relógios Circadianos/genética , Reparo do DNA , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
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
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
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
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
Doxorubicin (Dox) is an effective antineoplastic drug with serious cardiotoxic side effects that persist after drug withdrawal and can lead to heart failure. Dysregulation of vascular endothelium has been linked to the development of Dox-induced cardiotoxicity, but it is unclear whether and how transient exposure to Dox leads to long-term downregulation of Endothelial Vascular Endothelial Growth Factor Receptor type2 (VEGFR2), essential for endothelial cells function. Using an in vitro model devised to study the long-lasting effects of brief endothelial cells exposure to Dox, we show that Dox leads to sustained protein synthesis inhibition and VEGFR2 downregulation. Transient Dox treatment led to the development of long-term senescence associated with a reduction in VEGFR2 levels that persisted days after drug withdrawal. By analyzing VEGFR2 turnover, we ruled out that its downregulation was depended on Dox-induced autophagy. Conversely, Dox induced p53 expression, reduced mTOR-dependent translation, and inhibited global protein synthesis. Our data contribute to a mechanistic basis to the permanent damage caused to endothelial cells by short-term Dox treatment.
Assuntos
Regulação para Baixo , Doxorrubicina/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation.
Assuntos
Ácido Aspártico , Células Endoteliais , Alvo Mecanístico do Complexo 1 de Rapamicina , Neovascularização Patológica , Neovascularização Fisiológica , Animais , Ácido Aspártico/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/fisiologia , Biossíntese de Proteínas/fisiologia , Pirimidinas , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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
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
SAMHD1 is the major catabolic enzyme regulating the intracellular concentrations of DNA precursors (dNTPs). The S-phase kinase CDK2-cyclinA phosphorylates SAMHD1 at Thr-592. How this modification affects SAMHD1 function is highly debated. We investigated the role of endogenous SAMHD1 phosphorylation during the cell cycle. Thr-592 phosphorylation occurs first at the G1/S border and is removed during mitotic exit parallel with Thr-phosphorylations of most CDK1 targets. Differential sensitivity to the phosphatase inhibitor okadaic acid suggested different involvement of the PP1 and PP2 families dependent upon the time of the cell cycle. SAMHD1 turn-over indicates that Thr-592 phosphorylation does not cause rapid protein degradation. Furthermore, SAMHD1 influenced the size of the four dNTP pools independently of its phosphorylation. Our findings reveal that SAMHD1 is active during the entire cell cycle and performs an important regulatory role during S-phase by contributing with ribonucleotide reductase to maintain dNTP pool balance for proper DNA replication.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Fase S/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Treonina/química , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Ciclina A2/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Técnicas de Inativação de Genes , Humanos , Fosforilação , Proteólise , Ribonucleotídeo Redutases/metabolismo , Fase S/efeitos dos fármacos , Proteína 1 com Domínio SAM e Domínio HD/genética , Células THP-1RESUMO
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
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
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
Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.
Assuntos
Citosol/metabolismo , Mitocôndrias/metabolismo , Nucleotídeos de Timina/metabolismo , Animais , Ligação Competitiva , Transporte Biológico Ativo , Linhagem Celular , Replicação do DNA , DNA Mitocondrial/biossíntese , Humanos , Cinética , Camundongos , Modelos BiológicosRESUMO
We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159-12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine, and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol, whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of mdN and measure the transfer of radioactivity from [(3)H]thymidine to mt thymidine phosphates. In cycling cells lacking the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%, and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase.
Assuntos
5'-Nucleotidase/química , Desoxirribonucleotídeos/metabolismo , Ecdisterona/análogos & derivados , Mitocôndrias/metabolismo , Transporte Biológico , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , DNA/química , DNA/metabolismo , Relação Dose-Resposta a Droga , Ecdisterona/farmacologia , Inativação Gênica , Humanos , Mitocôndrias/enzimologia , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/química , Timidina Quinase/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.