Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).
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.
Ferraro
Paola
P
Department of Biology, The University of Padova, 35131 Padova, Italy.
Pontarin
Giovanna
G
Crocco
Laura
L
Fabris
Sonia
S
Reichard
Peter
P
Bianchi
Vera
V
D002460:Cell Line; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003470:Culture Media / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry*; D003857:Deoxyuridine / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology*; D017237:Mitochondrial Encephalomyopathies / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D013936:Thymidine / Q000737:chemistry; D013937:Thymidine Kinase / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000737:chemistry; D013997:Time Factors
D003470:Culture Media; D004272:DNA, Mitochondrial; D003854:Deoxyribonucleotides; D013939:Thymidine Phosphorylase; D013937:Thymidine Kinase; D013936:Thymidine; D003857:Deoxyuridine