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.     

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.     

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.     

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.     

Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency

Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.     

Synthesis of Mitochondrial DNA Precursors during Myogenesis,an Analysis in Purified C2C12 Myotubes

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.     

Loss of mitochondrial DNA under genotoxic stress conditions in the absence of the yeast DNA helicase Pif1p occurs independently of the DNA helicase Rrm3p

How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal conditions and in the presence of genotoxic stress is less understood. We demonstrate that the inefficient mtDNA replication process of mutant yeast cells lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA helicase RRM3. The rescue effect is likely due to the increase in the deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of genotoxic stress is not suppressed if RRM3 is lacking suggesting that this phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found to stimulate the incorporation of dNTPs into newly synthesised mtDNA of gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA helicase in mitochondria affects mtDNA replication directly. Possible roles of Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the temporarily displacement of tightly bound mtDNA-binding proteins, or the stabilization of the mitochondrial replication complex during mtDNA replication. X. Cheng, Y. Qin contributed equally to this work.     

Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

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.     

Tissue Specific Distribution of Pyrimidine Deoxynucleoside Salvage Enzymes Shed Light on the Mechanism of Mitochondrial DNA Depletion

Deficiency in thymidine kinase 2 (TK2) activity due to genetic alterations caused tissue specific mitochondrial DNA (mtDNA) depletion syndrome with symptoms resembling these of AIDS patients treated with nucleoside analogues. Mechanisms behind this mitochondrial effects is still not well understood. With rat as a model we isolated mitochondrial and cytosolic fractions from major organs and studied enzymes involved in thymidine (dT) and deoxycytidine (dC) phosphorylation by using ionic exchange column chromatography. A cytosolic form of TK2 was identified in all tested tissues in addition to mitochondrial TK2. TK1 was detected in liver and spleen cytosolic extracts while dCK was found in liver, spleen and lung cytosolic extracts. Thus, the nature of dT and dC salvage enzymes in each tissue type was determined. In most tissues TK2 is the only salvage enzyme present except liver and spleen. These results may help to explain the mechanisms of mitochondrial toxicity of antiviral nucleoside analogues and mtDNA depletion caused by TK2 deficiency.     

Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion

The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.     

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.     

Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.     

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.     

Ribonucleotide reductase subunit p53R2 regulates mitochondria homeostasis and function in KB and PC-3 cancer cells

Ribonucleotide reductase (RR) is a rate-limiting enzyme that catalyzes de novo conversion of ribonucleotide 5′-diphosphates to the corresponding 2′-deoxynucleotide, essential for DNA synthesis and replication. The mutations or knockout of RR small subunit, p53R2, results in the depletion of mitochondrial DNA (mtDNA) in human, implying that p53R2 might play a critical role for maintaining mitochondrial homeostasis. In this study, siRNA against p53R2 knockdown approach is utilized to examine the impact of p53R2 depletion on mitochondria and to derive underlying mechanism in KB and PC-3 cancer cells. Our results reveal that the p53R2 expression not only positively correlates with mtDNA content, but also partakes in the proper mitochondria function, such as ATP synthesis, cytochrome c oxidase activity and membrane potential maintenance. Furthermore, overexpression of p53R2 reduces intracellular ROS and protects the mitochondrial membrane potential against oxidative stress. Unexpectedly, knockdown of p53R2 has a modest, if any, effect on mitochondrial and total cellular dNTP pools. Taken together, our study provides functional evidence that mitochondria is one of p53R2-targeted organelles and suggests an unexpected function of p53R2, which is beyond known RR function on dNTP synthesis, in mitochondrial homeostatic control.