Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest kcat/Km values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

Free nucleotides in living cells play important roles in a variety of biological reactions, and often undergo chemical modifications of their base moieties. As modified nucleotides may have deleterious effects on cells, they must be eliminated from intracellular nucleotide pools. We have performed a screen for ITP‐binding proteins because ITP is a deaminated product of ATP, the most abundant nucleotide, and identified RS21‐C6 protein, which bound not only ITP but also ATP. Purified, recombinant RS21‐C6 hydrolyzed several canonical nucleoside triphosphates to the corresponding nucleoside monophosphates. The pyrophosphohydrolase activity of RS21‐C6 showed a preference for deoxynucleoside triphosphates and cytosine bases. The kcat/Km (s−1·m−1) values were 3.11 × 104, 4.49 × 103 and 1.87 × 103 for dCTP, dATP and dTTP, respectively, and RS21‐C6 did not hydrolyze dGTP. Of the base‐modified nucleotides analyzed, 5‐I‐dCTP showed an eightfold higher kcat/Km value compared with that of its corresponding unmodified nucleotide, dCTP. RS21‐C6 is expressed in both proliferating and non‐proliferating cells, and is localized to the cytoplasm. These results show that RS21‐C6 produces dCMP, an upstream precursor for the de novo synthesis of dTTP, by hydrolyzing canonical dCTP. Moreover, RS21‐C6 may also prevent inappropriate DNA methylation, DNA replication blocking or mutagenesis by hydrolyzing modified dCTP.

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Genomewide association studies have shown that a nonsynonymous single nucleotide polymorphism in PRKCH is associated with cerebral infarction and atherosclerosis-related complications. We examined the role of PKCg in lipid metabolism and atherosclerosis using apolipoprotein E-deficient (Apoe À/À ) mice. PKCg expression was augmented in the aortas of mice with atherosclerosis and exclusively detected in MOMA2-positive macrophages within atherosclerotic lesions. Prkch +/+ Apoe À/À and Prkch À/À Apoe À/À mice were fed a high-fat diet (HFD), and the dyslipidemia observed in Prkch +/+ Apoe À/À mice was improved in Prkch À/À Apoe À/À mice, with a particular reduction in serum LDL cholesterol and phospholipids. Liver steatosis, which developed in Prkch +/+ Apoe À/À mice, was improved in Prkch À/À Apoe À/À mice, but glucose tolerance, adipose tissue and body weight, and blood pressure were unchanged. Consistent with improvements in LDL cholesterol, atherosclerotic lesions were decreased in HFD-fed Prkch À/À Apoe À/À mice.Immunoreactivity against 3-nitrotyrosine in atherosclerotic lesions was dramatically decreased in Prkch À/À Apoe À/À mice, accompanied by decreased necrosis and apoptosis in the lesions.ARG2 mRNA and protein levels were significantly increased in Prkch À/À Apoe À/À macrophages.These data show that PKCg deficiency improves dyslipidemia and reduces susceptibility to atherosclerosis in Apoe À/À mice, showing that PKCg plays a role in atherosclerosis development.