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

doi:10.1074/jbc.m115.675082

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.

Highlights

Deoxyguanosine kinase deficiency causes mtDNA depletion in quiescent cells where SAMHD1 restricts dGTP concentration
To investigate possible functional interactions between the two enzymes, we employed skin fibroblasts derived from three unrelated mtDNA depletion syndrome patients [40,41,42] with different inactivating mutations in DGUOK, the gene coding for deoxyguanosine kinase, and compared them with WT human fibroblasts
The existence of the two mitochondrial deoxynucleoside kinases demonstrates that deoxynucleoside salvage is needed to support mtDNA synthesis under such conditions, but why are two distinct kinases required, and why inside mitochondria? At the end of S-phase thymidine kinase 1 (TK1) is degraded, the need for a second thymidine kinase is obvious, it may well reside outside mitochondria

Summary

Background

Deoxyguanosine kinase (dGK) deficiency causes mtDNA depletion in quiescent cells where SAMHD1 restricts dGTP concentration. 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. Downregulation of the enzyme by siRNA silencing, ubiquitin-dependent proteolysis, or genetic inactivation increases the concentrations of all dNTPs (9 –13) in human fibroblasts, especially that of dGTP [10] This effect is marked in quiescent or differentiated cells where the level of SAMHD1 is higher than during proliferation. Wishing to understand how SAMHD1 interacts with other enzymes in the regulation of dNTP pools, we set out to investigate its impact on the mt pools of cells with defective mt salvage of purine deoxyribonucleosides. We propose that inside mitochondria dGK is required to recycle the GdR released from extramitochondrial dGTP by SAMHD1

Experimental Procedures

Results

Discussion