The Experts below are selected from a list of 117 Experts worldwide ranked by ideXlab platform
Vera Bianchi - One of the best experts on this subject based on the ideXlab platform.
-
Unchanged Thymidine Triphosphate pools and Thymidine metabolism in two lines of Thymidine kinase 2-mutated fibroblasts.
The FEBS journal, 2009Co-Authors: Miriam Frangini, Chiara Rampazzo, Elisa Franzolin, M. C. Lara, Maya R. Vilà, Ramon Martí, Vera BianchiAbstract:Mitochondrial Thymidine kinase (TK2) catalyzes the phosphorylation of Thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and Thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, Thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [3H]Thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.
-
Mitochondrial Thymidine kinase and the enzymatic network regulating Thymidine Triphosphate pools in cultured human cells.
The Journal of biological chemistry, 2007Co-Authors: Chiara Rampazzo, Sonia Fabris, Elisa Franzolin, Katia Crovatto, Miriam Frangini, Vera BianchiAbstract:In non-proliferating cells mitochondrial (mt) Thymidine kinase (TK2) salvages Thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: Thymidine phosphorylase by degradation of Thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or Thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of Thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic Thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by Thymidine phosphorylase, which degrades Thymidine in the cytoplasm, thus limiting the availability of Thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of Thymidine concentrations at the nanomolar level.
-
Origins of mitochondrial Thymidine Triphosphate: Dynamic relations to cytosolic pools
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Giovanna Pontarin, Peter Reichard, Lisa Gallinaro, Paola Ferraro, Vera BianchiAbstract: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.
Guang Hsiung Kou - One of the best experts on this subject based on the ideXlab platform.
-
Chimeric polypeptide of Thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: Thymidine kinase activity of the recombinant protein expressed in a baculovirus/insect cell system.
Virology, 2002Co-Authors: Huey Fen Tzeng, Zee Fen Chang, Shao En Peng, Chung-hsiung Wang, Jung-yaw Lin, Guang Hsiung KouAbstract:Abstract The unique chimeric organization of the white spot syndrome virus (WSSV) tk-tmk gene encodes a protein which has significant homology to both cellular-type Thymidine kinase (TK) and cellular-type thymidylate kinase (TMK), but the functional activity of this protein has not been demonstrated. Because TK is usually expressed only at very low levels in host cells, in this study, the coding region of WSSV tk-tmk was expressed in an insect/baculovirus expression system. The His-tagged recombinant WSSV TK-TMK was purified by affinity chromatography, and its enzyme activity was characterized by steady-state kinetics. The recombinant WSSV TK-TMK catalyzed the phosphorylation of Thymidine to form Thymidine monophosphate (TMP), but we found no evidence that it was able to catalyze the further phosphorylation of TMP to form Thymidine diphosphate (or Thymidine Triphosphate). This TK activity is sensitive to feedback inhibition by Thymidine Triphosphate. In addition to Thymidine, of the nine other substrates tested, including acyclovir, ganciclovir, and 5-(2-bromovinyl)-2′-deoxyuridine, only 2′-deoxyuridine and 5-bromo-2′-deoxyuridine could also serve as substrates. These data suggest that the enzymatic characteristics of the recombinant WSSV TK-TMK are similar to those of the eukaryotic cytosolic TKs. We also found that TK activity increased as infection advanced in the integument and gills of experimentally infected shrimp, suggesting its functional involvement during WSSV infection.
Huey Fen Tzeng - One of the best experts on this subject based on the ideXlab platform.
-
Chimeric polypeptide of Thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: Thymidine kinase activity of the recombinant protein expressed in a baculovirus/insect cell system.
Virology, 2002Co-Authors: Huey Fen Tzeng, Zee Fen Chang, Shao En Peng, Chung-hsiung Wang, Jung-yaw Lin, Guang Hsiung KouAbstract:Abstract The unique chimeric organization of the white spot syndrome virus (WSSV) tk-tmk gene encodes a protein which has significant homology to both cellular-type Thymidine kinase (TK) and cellular-type thymidylate kinase (TMK), but the functional activity of this protein has not been demonstrated. Because TK is usually expressed only at very low levels in host cells, in this study, the coding region of WSSV tk-tmk was expressed in an insect/baculovirus expression system. The His-tagged recombinant WSSV TK-TMK was purified by affinity chromatography, and its enzyme activity was characterized by steady-state kinetics. The recombinant WSSV TK-TMK catalyzed the phosphorylation of Thymidine to form Thymidine monophosphate (TMP), but we found no evidence that it was able to catalyze the further phosphorylation of TMP to form Thymidine diphosphate (or Thymidine Triphosphate). This TK activity is sensitive to feedback inhibition by Thymidine Triphosphate. In addition to Thymidine, of the nine other substrates tested, including acyclovir, ganciclovir, and 5-(2-bromovinyl)-2′-deoxyuridine, only 2′-deoxyuridine and 5-bromo-2′-deoxyuridine could also serve as substrates. These data suggest that the enzymatic characteristics of the recombinant WSSV TK-TMK are similar to those of the eukaryotic cytosolic TKs. We also found that TK activity increased as infection advanced in the integument and gills of experimentally infected shrimp, suggesting its functional involvement during WSSV infection.
Miriam Frangini - One of the best experts on this subject based on the ideXlab platform.
-
Unchanged Thymidine Triphosphate pools and Thymidine metabolism in two lines of Thymidine kinase 2-mutated fibroblasts.
The FEBS journal, 2009Co-Authors: Miriam Frangini, Chiara Rampazzo, Elisa Franzolin, M. C. Lara, Maya R. Vilà, Ramon Martí, Vera BianchiAbstract:Mitochondrial Thymidine kinase (TK2) catalyzes the phosphorylation of Thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and Thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, Thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [3H]Thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.
-
Mitochondrial Thymidine kinase and the enzymatic network regulating Thymidine Triphosphate pools in cultured human cells.
The Journal of biological chemistry, 2007Co-Authors: Chiara Rampazzo, Sonia Fabris, Elisa Franzolin, Katia Crovatto, Miriam Frangini, Vera BianchiAbstract:In non-proliferating cells mitochondrial (mt) Thymidine kinase (TK2) salvages Thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: Thymidine phosphorylase by degradation of Thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or Thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of Thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic Thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by Thymidine phosphorylase, which degrades Thymidine in the cytoplasm, thus limiting the availability of Thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of Thymidine concentrations at the nanomolar level.
Zee Fen Chang - One of the best experts on this subject based on the ideXlab platform.
-
Chimeric polypeptide of Thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: Thymidine kinase activity of the recombinant protein expressed in a baculovirus/insect cell system.
Virology, 2002Co-Authors: Huey Fen Tzeng, Zee Fen Chang, Shao En Peng, Chung-hsiung Wang, Jung-yaw Lin, Guang Hsiung KouAbstract:Abstract The unique chimeric organization of the white spot syndrome virus (WSSV) tk-tmk gene encodes a protein which has significant homology to both cellular-type Thymidine kinase (TK) and cellular-type thymidylate kinase (TMK), but the functional activity of this protein has not been demonstrated. Because TK is usually expressed only at very low levels in host cells, in this study, the coding region of WSSV tk-tmk was expressed in an insect/baculovirus expression system. The His-tagged recombinant WSSV TK-TMK was purified by affinity chromatography, and its enzyme activity was characterized by steady-state kinetics. The recombinant WSSV TK-TMK catalyzed the phosphorylation of Thymidine to form Thymidine monophosphate (TMP), but we found no evidence that it was able to catalyze the further phosphorylation of TMP to form Thymidine diphosphate (or Thymidine Triphosphate). This TK activity is sensitive to feedback inhibition by Thymidine Triphosphate. In addition to Thymidine, of the nine other substrates tested, including acyclovir, ganciclovir, and 5-(2-bromovinyl)-2′-deoxyuridine, only 2′-deoxyuridine and 5-bromo-2′-deoxyuridine could also serve as substrates. These data suggest that the enzymatic characteristics of the recombinant WSSV TK-TMK are similar to those of the eukaryotic cytosolic TKs. We also found that TK activity increased as infection advanced in the integument and gills of experimentally infected shrimp, suggesting its functional involvement during WSSV infection.