BBRC Biochemical and Biophysical Research Communications 310 (2003) 963–966 www.elsevier.com/locate/ybbrc
Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency Ann Saada,a,* Efrat Ben-Shalom,a Rivka Zyslin,a Chaya Miller,a Hanna Mandel,b and Orly Elpelega,c a
Metabolic Disease Unit, Shaare-Zedek Medical Center, Jerusalem, Israel b Department of Pediatrics, Rambam Medical Center, Haifa, Israel c Hebrew University Faculty of Medicine, Jerusalem, Israel Received 7 August 2003
Abstract 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. Ó 2003 Elsevier Inc. All rights reserved. Keywords: mtDNA depletion; Thymidine kinase 2
The mitochondria harbor a separate genome, a double stranded, 16.5 kb, circular molecule whose replication is not cell cycle dependent. Mitochondrial DNA (mtDNA) synthesis requires the import of cytosolic deoxynucleosides (dNPs) through dedicated transporters or salvaging deoxynucleosides within the mitochondrial matrix. Apparently, enzymes of the de novo dNTP synthesis pathway are not present in the mitochondria. A defect in the mitochondrial salvage system would therefore remain subtle in replicating cells, where cytosolic dNTPs are continuously synthesized and would mainly affect senescent tissues [1–3]. In agreement, we and others have recently reported a defect in the mitochondrial TK (TK2) which manifested with isolated skeletal myopathy and mtDNA depletion in muscle but not in skin and blood [4–7]. TK2 catalyzes the first rate limiting step of the mitochondrial salvage pathway, phosphorylating deoxythymidine (dThd), deoxycytidine (dCyt), and dUrd [8]. We hypothesized that in replicating cells cytosolic
TK1, dCK, and the de novo nucleotide synthesis pathway would compensate for the defect in TK2 activity. In senescent cells, however, a shortage of mitochondrial dTTP is expected as a result of downregulated TK1 and ribonucleotide reductase activities [3,8,9]. In order to elucidate the pathomechanism of TK2 deficiency we measured mitochondrial dNTP pools in non-replicating fibroblasts from patients with muscle mtDNA depletion due to mutations in TK2.
Patients, materials, and methods Patients Fibroblast cell lines from two patients with muscle mtDNA depletion due to TK2 deficiency were included: patient 1 was homozygous for an Ile181Asn mutation and patient 2 homozygous for a His90Asn mutation in the TK2 gene [4]. No other tissues were available from the patients. Materials
* Corresponding author. Fax: +972-2-652-3114. E-mail address:
[email protected] (A. Saada-Reisch).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.104
Tissue culture medium, supplements, and E-Z PCR Mycoplasma Test Kit were purchased from Biological Industries (Beth Haemek,
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Israel). Radioactive deoxyribonucleotides for TK2 and dNTP determination were purchased from Amersham–Pharmacia Biotech UK (Buckinghamshire, UK). DNA polymerase I Klenow fragment was purchased from New England Biolabs (Beverly, MA). Primers were from Sigma Genosys (Rehovot, Israel) All other chemicals were from Sigma Chemical (St. Louis, MO). Methods Tissue cultures. Fibroblast cell line was established from patient and control forearm biopsies performed with informed consent. The cells were grown in Dulbecco’s minimal essential medium containing 4.5 g/L glucose, supplemented with 10% fetal calf serum, 50 lg/ml uridine, 110 lg/ml pyruvate at 37 °C in an atmosphere of 5% CO2 . Fibroblasts were maintained at least for a week after reaching confluence medium was changed to medium lacking uridine, and pyruvate was supplemented with 10% dialyzed fetal calf serum 24 h prior to mitochondrial isolation. Tissue cultures were screened for mycoplasma contamination by the EZ-PCR Mycoplasma Test Kit and were found to be negative. Mitochondrial preparations. For enzymatic studies, fibroblast mitochondria were isolated by nitrogen cavitation and centrifugation as we have previously described [9]. For the measurement of dNTP pools mitochondria were isolated using digitonin with the emphasis on speed rather than purity; The mitochondrial isolation and nucleotide extraction methods were modified from Bestwich et al. [10] and Gallinario et al. [11]. Fibroblasts from at least three T75 flasks were washed in phosphate-buffered saline trypsinized at room temperature and viable cells were counted by trypan blue exclusion. The following steps were performed at 0 °C: fibroblast suspended to a concentration of 8 106 cells/ml in a buffer containing 210 mM mannitol, 70 mM sucrose, 0.2 mM ethylene glycol bis aminoethyl ether tetraacetic acid, and 10 mM Tris at pH 7.5 with the addition of 2 mg/ml digitonin. The suspension was passed through a 21 gauge needle 10 times diluted in buffer lacking digitonin and the suspension was centrifuged at 800g for 5 min to remove debris and nuclei. Subsequently the supernatant was centrifuged at 18,000g for 15 min in order to obtain mitochondria. Nucleotides were extracted from mitochondrial pellet by 60% methanol for 30 min on ice. After a heating step and centrifugation the supernatant was evaporated by lyophilization and the lyophilized pellet was stored at )70 °C until determination of dNTP pools [11]. In order to calculate the mitochondrial yield in each preparation, citrate synthase (CS) activity was determined in the homogenate and mitochondrial pellet. A typical preparation had a yield around approximately 50%. Enzyme assays. TK2 activity in isolated mitochondria was determined with 11 lM [3 H]dThd or [3 H]dCyt as substrates as described previously [9,12]. Citrate synthase and cytochrome c oxidase (COX) were performed using standard spectrophotometric assays [13,14]. dNTP pool determination. dNTPs in mitochondrial extracts of the patients and six controls were quantitated by an enzymatic assay using synthetic oligonucleotides as template primers according to Sherman
and Fyfe [15] with the following modifications: the reaction mixture was 25 ll containing 100 mM Hepes, pH 7.3, 10 mM MgCl2 with 0.125 U DNA polymerase I Klenow fragment, 2.5 lM [3 H]dATP or [3 H]dTTP (for dATP determinations), primer template, and 5 ll standard or sample. For dTTP and dATP determinations 0.25 lM template primer was used while for dCTP and dGTP 0.05 lM template primer was optimal. After incubation at 25 °C for 1 h the reaction was spotted on DE81 filters. After washing the filters were treated with 2 M NaOH for 30 min and liquid scintillation cocktail overnight to minimize quenching. The dNTP contents were calculated as pmol/106 cells with correction for incomplete mitochondrial yield. Determination of mtDNA: nuclear DNA ratio. Total DNA extracted from fibroblasts, digested by PvuII, was hybridized to one 18S rRNA probe and a mixture of three mtDNA probes as previously described. Signals were quantified by phosphorimaging and the mtDNA/nuclear DNA ratio was calculated [4].
Results The results of the determination of the activity of TK2, COX, and CS in the patients’ fibroblasts are presented in Table 1. The activity of TK2 in fibroblast mitochondria with dThd as a substrate was reduced to 53% and 47% of the control for patients 1 and 2, respectively. Using dCyt as a substrate, the decrease in TK2 activity was more pronounced, 5% and 31% of the control mean, in patients 1 and 2, respectively. The activity of COX, normalized for CS activity, and the mtDNA to nuclear nDNA ratio were within the normal control range in both patients. In order to accurately determine the dNTP mitochondrial pools a standard curve was constructed for each dNTP. Linear correlation was obtained between radioactivity and the amount of dNTP within a range of 0.1–0.5 pmol per reaction (Fig. 1). In the patients’ mitochondria, dTTP content was most severely affected, being decreased to 48% and 29% of the control, in patients 1 and 2, respectively. Pool size of the other TK2 product, dCTP, was lesser affected, being reduced to 65% and 59% of the control mean in patients 1 and 2, respectively. dATP was similarly decreased while dGTP was normal in patient 1 and mildly decreased in patient 2 (Table 2). Calculated deoxypyrimidine triphosphate:deoxypurine triphosphate (pyr:pur) ratio was 57% and 69% of the control while the dTTP:dCTP ratio 73% and 48% in patients 1 and 2, respectively.
Table 1 Enzymatic activities of COX, TK2, and mtDNA/nDNA ratio Fibroblasts
Patient 1 Patient 2 Controls, n ¼ 7
COX (nmol/min/mg)
369 217 394 143
COX/CS ratio
1.65 1.23 1.71 0.51
TK2 (pmol/min/mg) dThd
dCyt
40 36 76 22
2.3 14.4 46.0 12
mtDNA/nDNA ratio
1.2 1.5 1.3 0.3
Cytochrome c oxidase (COX), citrate synthase (CS) activities, and thymidine kinase 2 (TK2) with dThd and dCyt as substrates were determined in mitochondria isolated from patients and control fibroblasts. The enzymatic activities for patients are expressed as mean values from at least two mitochondrial preparations. The mtDNA/nuclear DNA (nDNA) ratios were determined from total fibroblast DNA.
A. Saada et al. / Biochemical and Biophysical Research Communications 310 (2003) 963–966
Fig. 1. Standard curves for dNTP determination. Standard curves were constructed for each deoxyribonucloeside triphosphate using the enzymatic assay with synthetic oligonucleotides as template primers. Examples of typical standard curves are presented.
Table 2 Fibroblast mitochondrial dNTP content Fibroblasts
dTTP
dCTP
dGTP
dATP
Patient 1 Patient 2 Controls, n¼6
0.53 0.32 1.11 0.25
0.81 0.73 1.24 0.33
1.89 1.16 1.68 0.21
0.57 0.45 0.80 0.12
dNTP pools were determined in patient and control fibroblast mitochondria. All values are expressed as pmol mitochondrial deoxyribonucleotide triphosphate/106 cells. Patient values are means of at least two mitochondrial extracts with duplicate determinations of each pool.
Discussion Based on the phenotypic heterogeneity associated with mtDNA depletion, it is clear that this molecular abnormality is the end result of defects in any of several proteins, most of which are yet to be identified. The recent findings of mutations in the TK2 gene in a number of patients with fatal infantile skeletal myopathy underscored the indispensability of the deoxyribonucleoside salvage pathway for mtDNA synthesis [4–7]. A similarly essential role was shown for thymidine phosphorylase and deoxyguanosine kinase [16,17]. These discoveries have raised the question of the exact sequence of events leading to mtDNA depletion. In the present study we set to investigate the effect of TK2 deficiency on the mitochondrial dNTP pools. Care was taken to use confluent, non-replicating cells in order to minimize the cytosolic contribution to mitochondrial dNTP pools [8,18]. Using these measures, mitochondrial dNTP pool sizes in our control fibroblast were in the same order of magnitude as published for HeLa cells [10]. The variability in the relative contribution of each dNTP to the total pool likely reflects differences in growth phase and cell type [18].
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We have previously speculated a decreased pyr:pur ratio in TK2 deficient patients’ tissues and this has now been verified [2,4]. We could not predict, however, the exact aberration of the mitochondrial dCTP:dTTP ratio; since normal TK2 exhibits higher efficiency with dThd than with dCyt, mutated TK2 may reduce the dCTP:dTTP ratio even further [12]. However, dCK, the cytosolic enzyme which phosphorylates dCyt throughout the cell cycle, could modulate the dCTP:dTTP ratio in the opposite direction, bringing it back to nearly normal levels [8]. Our finding that dTTP content is the most severely affected dNTP and the lowered dTTP:dCTP ratio suggests that in non-replicating cells of patients with TK2 deficiency, dCK contribution is more significant than the differential efficiency of TK2 towards its substrates. We speculate that the reduced dATP content in both patients is the result of downregulated deoxyadenosine phosphorylation in response to reduced dTTP content, possibly in an attempt to normalize the dTTP:dATP ratio. The fact that dNTP aberrations in our patients’ cells are mild, enabling mtDNA synthesis to continue normally, is attributed to the considerable residual TK2 activity with dThd as a substrate. However, this should not discredit our findings which can still be used for illustrating the tendency of mitochondrial dNTP aberrations in more severely affected, non-replicating tissues such as muscle; we have previously shown that TK2 activity in control muscle is about 15% of the activity in control fibroblasts [9]. We therefore anticipate a more pronounced reduction of mitochondrial dTTP content and a markedly lower pyr:pur ratio in patients’ muscle tissue. dNTP imbalance has been associated with an increased mutation rate, breakage of mature DNA, and inhibition of its repair [19,20]. The finding that mtDNA depletion is the only molecular abnormality in TK2 deficient muscle is therefore puzzling. We have previously proposed that the absence of mutations in mtDNA in our patients’ muscle is the result of wellbalanced mitochondrial dNTP pools in fetal life as all tissues might be regarded as replicating. In addition, increased fidelity due to the slowdown of the DNA polymerase activity has been associated with decreased dNTP content [21]. Finally the determination of mitochondrial dNTP pools size is emerging as an invaluable tool in the elucidation of the pathophysiology of quantitative mtDNA defects and may turn to be of major help in future therapy design in these disorders. Acknowledgments Drs. Vera Bianchi, Emma Mansson, and Michio Hirano are acknowledged for helpful discussions. Yaron Shoshani is acknowledged for excellent technical assistance. This study was funded by the Israeli
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Academy of Sciences and Humanities, Grant No. 406/01-1, and the Israeli Ministry of Health Grant No. 5307.
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