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

Elisa Franzolin, Cristiano Salata, Vera Bianchi, Chiara Rampazzo, Elisa Franzolin, Cristiano Salata, Vera Bianchi, Chiara Rampazzo

Abstract

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.

Keywords: dGTP pool; gene silencing; mitochondrial DNA (mtDNA); mitochondrial deoxynucleoside salvage; mitochondrial disease; mtDNA precursors; nucleoside/nucleotide metabolism; phosphatase; ribonucleotide reductase.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.
FIGURE 1.
Interaction between SAMHD1 and deoxyguanosine kinase activities in the regulation of mitochondrial dGTP. Synthesis of dGTP occurs in the cytosol by de novo synthesis catalyzed by RNR and by salvage of GdR catalyzed by dCK. The two synthetic pathways are counteracted by catabolic activities distributed between the nucleus and cytoplasm. SAMHD1 degrades dGTP to GdR in the nucleus, whereas in the cytosol 5′-nucleotidases (cdN and cN-II) dephosphorylate dGMP to GdR, and PNP degrades GdR to free guanine. Due to the permeability of the nuclear envelope to nucleotides and nucleosides, nuclear and cytosolic pools represent a single kinetic compartment. The mitochondrial dGTP pool derives most of its components from the cytosol via nucleotide and nucleoside carriers in the inner mt membrane. In mitochondria, a separate salvage pathway, depending on dGK, recycles GdR, creating a dGTP pool protected from the catabolic activity of SAMHD1.
FIGURE 2.
FIGURE 2.
mtDNA copy number and mitochondrial mass in WT and dGK-mutated fibroblasts.A, we measured mtDNA copy number per nuclear genome in proliferating (48 and 72 h) and quiescent cultures of three lines of wt skin fibroblasts (C1, C2, C3) and three dGK-mutated lines (P1, P2, P3). Control values were combined (wt). All data are the means ± S.E. from at least two experiments for each cell line analyzed in triplicate. B, mitochondrial mass was evaluated by measuring citrate synthase activity (units (U) = nmol/min) in whole cell extracts and by normalizing the nonyl acridine orange (NAO) fluorescence of each line determined by flow cytometry by that of control C1. Data are the means ± S.E. from two experiments.
FIGURE 3.
FIGURE 3.
Cytosolic and mitochondrial dNTP pool sizes in quiescent skin fibroblasts. Cytosolic (A) and mitochondrial (B) dNTP pools were measured in quiescent cultures of two WT (C1, C2) and three dGK-mutated (P1, P2, P3) lines of skin fibroblasts. To compare the two groups we calculated the mean ratios between the size of each dNTP pool and that of dGTP in the cytosol (C) and mitochondria (D) of WT or dGK-mutated fibroblasts. Data are the means ± S.E. from two experiments for each cell line analyzed in duplicate. Asterisks indicate a significant difference (***, p < 0.001) of the dCTP/dGTP ratio in mutant fibroblasts relative to controls.
FIGURE 4.
FIGURE 4.
Expression of ribonucleotide reductase subunits and SAMHD1 in untreated quiescent skin fibroblasts. Ribonucleotide reductase subunits (R2, p53R2, and R1) and SAMHD1 were detected by immunoblotting in extracts from one proliferating WT control culture (C1p) and quiescent cultures of three WT (C1, C2, and C3) and three dGK-mutated (P1, P2, and P3) lines of skin fibroblasts. To compare R2 or R1 in proliferating and quiescent extracts, different amounts of proteins were loaded in the respective lanes of the gels: 4 and 40 μg for R2 and 20 and 40 μg for R1. In the case of p53R2 we used 2 μg of quiescent extracts and 20 μg for SAMHD1. Loading controls for R1, R2, and SAMHD1 were two unspecific bands (asterisks), whereas GAPDH was used as the control for p53R2.
FIGURE 5.
FIGURE 5.
Effect of SAMHD1 silencing on mtDNA content of quiescent skin fibroblasts. mtDNA copy number normalized per nuclear genome was determined in WT and dGK-mutated fibroblasts at confluence, i.e. before starting the transfections with siRNAs in low serum medium and after 10 days of transfection during quiescence with control siRNA or anti-SAMHD1 siRNA. The values of three WT lines (C1, C2, and C3) were averaged, whereas those of the patient lines (P1, P2, P3) are reported separately. The percent residual level of SAMHD1 mRNA in the silenced cultures is indicated. Data are the means ± S.E. from three to six experiments for each cell line analyzed in triplicate.
FIGURE 6.
FIGURE 6.
Quantitative variations of mtDNA during siRNA silencing of SAMHD1 in quiescent WT and dGK-mutated fibroblasts.A, expression of SAMHD1 protein during transfection with control or anti-SAMHD1 siRNA in quiescent cultures of WT (C2) or dGK-mutated (P1, P2) fibroblasts. The arrow indicates the SAMHD1 signal. An unspecific band appeared just above SAMHD1 and remained stable during silencing. B, residual level of SAMHD1 mRNA in the silenced cultures relative to that in the cultures transfected with control siRNA was determined at the indicated days of transfection during quiescence. Red, C2; green, P1; blue, P2. C, mtDNA content per nuclear genome was measured at 1- or 2-day intervals in cultures treated with control siRNA (continuous line) or anti-SAMHD1 siRNA (broken line) during 10 days of quiescence. Color coding is in B.
FIGURE 7.
FIGURE 7.
Effects of SAMHD1 silencing on cytosolic and mitochondrial dNTP pools in quiescent WT and dGK-mutated fibroblasts. Shown is the increase of cytosolic (A) and mitochondrial (B) dNTP pools induced by SAMHD1 silencing in WT and dGK-mutated skin fibroblasts incubated during 10 days of quiescence with anti-SAMHD1 siRNA or control siRNA. The increases measured in three WT lines were averaged as well as those of the P2 and P3 mutant lines. Values of the P1 mutant are reported separately. C, percentage of dGTP relative to the total dNTPs in the cytosolic and mt pools in WT and dGK-mutated fibroblasts transfected with control siRNA (black) or with anti-SAMHD1 siRNA (gray). D and E, composition of cytosolic (D) and mitochondrial (E) dNTP pools in cultures transfected with control siRNA or with anti-SAMHD1 siRNA, expressed as ratios between the sizes of the individual dNTP pools and the corresponding dGTP pool. The ratios of WT cells were averaged. Mutant P1 values are reported separately from those of the other two mutants (P2/P3). Data are the means ± S.E. from two experiments for each cell line analyzed in duplicate. Asterisks indicate that in nonsilenced cultures significant differences exist in the dTTP/dGTP and dCTP/dGTP ratios of dGK-mutated fibroblasts (P1, P2/P3) relative to controls (wt). **, p < 0.01; *, p < 0.05.

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