Mitochondrial purine and pyrimidine metabolism and beyond

ABSTRACT

Carefully balanced deoxynucleoside triphosphate (dNTP) pools are essential for both nuclear and mitochondrial genome replication and repair. Two synthetic pathways operate in cells to produce dNTPs, e.g., the de novo and the salvage pathways. The key regulatory enzymes for de novo synthesis are ribonucleotide reductase (RNR) and thymidylate synthase (TS), and this process is considered to be cytosolic. The salvage pathway operates both in the cytosol (TK1 and dCK) and the mitochondria (TK2 and dGK). Mitochondrial dNTP pools are separated from the cytosolic ones owing to the double membrane structure of the mitochondria, and are formed by the salvage enzymes TK2 and dGK together with NMPKs and NDPK in postmitotic tissues, while in proliferating cells the mitochondrial dNTPs are mainly imported from the cytosol produced by the cytosolic pathways. Imbalanced mitochondrial dNTP pools lead to mtDNA depletion and/or deletions resulting in serious mitochondrial diseases. The mtDNA depletion syndrome is caused by deficiencies not only in enzymes in dNTP synthesis (TK2, dGK, p53R2, and TP) and mtDNA replication (mtDNA polymerase and twinkle helicase), but also in enzymes in other metabolic pathways such as SUCLA2 and SUCLG1, ABAT and MPV17. Basic questions are why defects in these enzymes affect dNTP synthesis and how important is mitochondrial nucleotide synthesis in the whole cell/organism perspective? This review will focus on recent studies on purine and pyrimidine metabolism, which have revealed several important links that connect mitochondrial nucleotide metabolism with amino acids, glucose, and fatty acid metabolism.     

Deoxyribonucleoside kinases in mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.     

Deoxyribonucleoside Kinases in Mitochondrial DNA Depletion

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non ‐replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible toTK2 deficiency. The precise patho‐physiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Molecular Mechanisms of Mitochondrial DNA Depletion Diseases Caused by Deficiencies in Enzymes in Purine and Pyrimidine Metabolism

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.     

Molecular mechanisms of mitochondrial DNA depletion diseases caused by deficiencies in enzymes in purine and pyrimidine metabolism

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.     

Down-regulation of mitochondrial thymidine kinase 2 and deoxyguanosine kinase by didanosine: Implication for mitochondrial toxicities of anti-HIV nucleoside analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial rate limiting phosphorylation of deoxynucleosides and are essential enzymes for mitochondrial function. Chemotherapy using nucleoside analogs is often associated with mitochondrial toxicities. Here we showed that incubation of U2OS cells with didanosine (ddI, 2′,3′-dideoxyinosine), a purine nucleoside analog used in the highly active antiretroviral therapy (HAART), led to selective degradation of both mitochondrial TK2 and dGK while the cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) were not affected. Addition of guanosine to the ddI-treated cells prevented the degradation of mitochondrial TK2 and dGK. The levels of intracellular reactive oxygen species and protein oxidation in ddI-treated and control cells were also measured. The results suggest that down-regulation of mitochondrial TK2 and dGK may be a mechanism of mitochondrial toxicity caused by antiviral and anticancer nucleoside analogs.     

A mathematical model of human thymidine kinase 2 activity

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ～0.5 (apparent negative cooperativity) for dT and ～1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.     

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids.

Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.     

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.     

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.     

Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease with mitochondrial DNA (mtDNA) alterations and is caused by mutations in the nuclear gene encoding thymidine phosphorylase (TP). The cardinal clinical manifestations are ptosis, ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Skeletal muscle shows mitochondrial abnormalities, including ragged-red fibers and cytochrome c oxidase deficiency, together with mtDNA depletion, multiple deletions or both. In MNGIE patients, TP mutations cause a loss-of-function of the cytosolic enzyme, TP. As a direct consequence of the TP defect, thymidine metabolism is altered. High blood levels of this nucleoside are likely to lead to mtDNA defects even in cells that do not express TP, such as skeletal muscle. We hypothesize that high concentrations of thymidine affect dNTP (deoxyribonucleoside triphosphate) metabolism in mitochondria more than in cytosol or nuclei, because mitochondrial dNTPs depend mainly on the thymidine salvage pathway, whereas nuclear dNTPs depend mostly on de novo pathway. The imbalance in the mitochondrial dNTP homeostasis affects mtDNA replication, leading to mitochondrial dysfunction.     

Substrate Specificities,Expression and Primary Sequences of Deoxynucleoside Kinases; Implications for Chemotherapy

Abstract

Deoxynucleoside kinases are key enzyme in deoxyribonucleoside salvage, phosphorylating many important anti cancer and anti viral drugs. There are four kinases in animal cells; cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The biochemical properties of the purified enzymes and the sequences of their cDNA;s have been determined. In case of TK2 and dGK this was done very recently and they show high homology to dCK and the herpes virus kinases but not to TK1. The evolutionary and functional consequences of this fact will be discussed.     

The Role of Mitochondrial dNTP Levels in Cells with Reduced TK2 Activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

The role of mitochondrial dNTP levels in cells with reduced TK2 activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

Thymidine phosphorylase deficiency causes MNGIE: an autosomal recessive mitochondrial disorder

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site-specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.     

Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site‐specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.     

Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.