title: Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.
abstract: 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 [(3)H]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.
authors.affiliation: Department of Biology, University of Padova, Italy.
authors.forename: Miriam
authors.initials: M
authors.lastname: Frangini
authors.forename: Chiara
authors.initials: C
authors.lastname: Rampazzo
authors.forename: Elisa
authors.initials: E
authors.lastname: Franzolin
authors.forename: Mari-Carmen
authors.initials: MC
authors.lastname: Lara
authors.forename: Maya R
authors.initials: MR
authors.lastname: Vilà
authors.forename: Ramon
authors.initials: R
authors.lastname: Martí
authors.forename: Vera
authors.initials: V
authors.lastname: Bianchi
mesh_terms: D000293:Adolescent; D002478:Cells, Cultured; D002648:Child; D003600:Cytosol / Q000201:enzymology; D003842:Deoxycytidine Kinase / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009708:Nucleotidases / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D013939:Thymidine Phosphorylase / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D021122:Protein Subunits; D013942:Thymine Nucleotides; D012264:Ribonucleotide Reductases; D013939:Thymidine Phosphorylase; C105339:thymidine kinase 2; D013937:Thymidine Kinase; D003842:Deoxycytidine Kinase; D009708:Nucleotidases; C028331:deoxynucleotidase; C024157:thymidine 5'-triphosphate; D013936:Thymidine