Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2

Abstract

Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

“…Intriguingly, 2 of these variants both affect residue Arg139 that is located in the α helix a2 at the base of the substrate binding pocket of NUDT15 52 . It has been hypothesized that the substitution of arginine with cysteine (p.Arg139Cys) at this position might introduce a disulfide bond and thus structural perturbation that interferes with TGTP binding 52 . In contrast, the arginine-to-histidine change caused by the p.Arg139His variant might lead to a reduced eletrophilicity and compromise substrate interaction.…”

“…However, this was challenged by more recent data showing a strong preference of NUDT15 for thiopurine metabolites over oxo-dGTP 52 . NUDT15 is also shown to efficiently hydrolyze dGTP with potential importance in purine nucleotide homeostasis 52 , but its exact physiological functions remain unknown. It is unclear whether loss-of-function NUDT15 variants confer susceptibility to any diseases, although there is no evidence in the literature to suggest that this is the case.…”

Oxidative stress and DNA damage induced by spinosad exposure in Spodoptera frugiperda Sf9 cells

“…The OGG1 and MTH1 proteins are well-studied repair enzymes that hydrolyses oxidized bases, thus preventing their incorporation into DNA (Carter et al, 2015). Treatment of cells with spinosad resulted in a time-dependent accumulation of OGG1 and MTH1 protein, Figure 2.…”

Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2ʹ-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/ MTH2 Expression in Hepatocytes

“…The activity of MTH2 towards 8-oxo-dGTP has led to the assumption that it may contribute to sanitizing the oxidized dNTP pool despite much lower activity than MTH1 towards 8-oxo-dGTP or 2-OH-dATP [23,60]. More recently, Carter et al reported that MTH2 was structurally distinct from MTH1 and had considerably lower activity to oxidized guanine species than to the undamaged nucleotides [61]. Moreover, knockdown of MTH2 did not alter the level of 8-oxodG incorporation into DNA or cancer cell survival, suggesting that MTH2 might not sanitize 8-oxo-dGTP in the cells [61].…”

“…More recently, Carter et al reported that MTH2 was structurally distinct from MTH1 and had considerably lower activity to oxidized guanine species than to the undamaged nucleotides [61]. Moreover, knockdown of MTH2 did not alter the level of 8-oxodG incorporation into DNA or cancer cell survival, suggesting that MTH2 might not sanitize 8-oxo-dGTP in the cells [61]. Regardless, a most recent study using CRISPR/Cas9 approach to knock out MTH1, MTH2 or both in HeLa S3 cells demonstrated an increased sensitivity to hydrogen peroxide whereas overproduction of MTH1 and MTH2 almost completely suppressed the mutator phenotype of mutT-deficient cells, suggesting that both MTH1 and MTH2 are involved in the maintaining genome stability in human cells against oxidative stress [62].…”