Sanitation enzymes: Exquisite surveillance of the noncanonical nucleotide pool to safeguard the genetic blueprint.

Reactive oxygen species (ROS) are common products of normal cellular metabolism, but their elevated levels can result in nucleotide modifications. These modified or noncanonical nucleotides often integrate into nascent DNA during replication, causing lesions that trigger DNA repair mechanisms such as the mismatch repair machinery and base excision repair. Four superfamilies of sanitization enzymes can effectively hydrolyze noncanonical nucleotides from the precursor pool and eliminate their unintended incorporation into DNA. Notably, we focus on the representative MTH1 NUDIX hydrolase, whose enzymatic activity is ostensibly nonessential under normal physiological conditions. Yet, the sanitization attributes of MTH1 are more prevalent when ROS levels are abnormally high in cancer cells, rendering MTH1 an interesting target for developing anticancer treatments. We discuss multiple MTH1 inhibitory strategies that have emerged in recent years, and the potential of NUDIX hydrolases as plausible targets for the development of anticancer therapeutics.

NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

Nudix hydrolase 14 influences plant development and grain chalkiness in rice.

Nudix hydrolases (NUDX) can hydrolyze a wide range of organic pyrophosphates and are widely distributed in various organisms. Previous studies have shown that NUDXs are extensively involved in biotic and abiotic stress responses in different plant species; however, the role of NUDXs in plant growth and development remains largely unknown. In the present study, we identified and characterized OsNUDX14 localized in the mitochondria in rice. Results showed that OsNUDX14 is constitutively expressed in various tissues and most strongly expressed in mature leaves. We used CRISPR/Cas9 introducing mutations that editing OsNUDX14 and its encoding product. OsNUDX14-Cas9 (nudx14) lines presented early flowering and a larger flag leaf angle during the reproductive stage. In addition, OsNUDX14 affected grain chalkiness in rice. Furthermore, transcript profile analysis indicated that OsNUDX14 is associated with lignin biosynthesis in rice. Six major haplotypes were identified by six OsNUDX14 missense mutations, including Hap_1 to Hap_6. Accessions having the Hap_5 allele were geographically located mainly in South and Southeast Asia with a low frequency in the Xian/indica subspecies. This study revealed that OsNUDX14 is associated with plant development and grain chalkiness, providing a potential opportunity to optimize plant architecture and quality for crop breeding.

Structural Insight into Molecular Inhibitory Mechanism of InsP6 on African Swine Fever Virus mRNA-Decapping Enzyme g5Rp.

Removal of 5' cap on cellular mRNAs by the African swine fever virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate-linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and the hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer is composed of a unique N-terminal helical domain and a C-terminal classic Nudix domain. As g5Rp is an mRNA-decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp which are important to RNA binding and decapping enzyme activity. Furthermore, the g5Rp-mediated mRNA decapping was inhibited by InsP6. The g5Rp-InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp. IMPORTANCE ASF is a highly contagious hemorrhagic viral disease in domestic pigs which causes high mortality. Currently, there are still no effective vaccines or specific drugs available against this particular virus. The protein g5Rp is the only viral mRNA-decapping enzyme, playing an essential role in the machinery assembly of mRNA regulation and translation initiation. In this study, we solved the crystal structures of g5Rp dimer and complex with InsP6. Structure-based mutagenesis studies revealed critical residues involved in a candidate RNA binding region, which also play pivotal roles in complex with InsP6. Notably, InsP6 can inhibit g5Rp activity by competitively blocking the binding of substrate mRNA to the enzyme. Our structure-function studies provide the basis for potential anti-ASFV inhibitor designs targeting the critical enzyme.

Studies on the Regulation of (p)ppGpp Metabolism and Its Perturbation Through the Over-Expression of Nudix Hydrolases in Escherichia coli.

Stringent response mediated by modified guanosine nucleotides is conserved across bacteria and is regulated through the Rel/Spo functions. In Escherichia coli, RelA and SpoT proteins synthesize the modified nucleotides ppGpp and pppGpp, together referred to as (p)ppGpp. SpoT is also the primary (p)ppGpp hydrolase. In this study, using hypomorphic relA alleles, we provide experimental evidence for SpoT-mediated negative regulation of the amplification of RelA-dependent stringent response. We investigated the kinetics of ppGpp degradation in cells recovering from stringent response in the complete absence of SpoT function. We found that, although greatly diminished, there was slow ppGpp degradation and growth resumption after a lag period, concomitant with decrease in ppGpp pool. We present evidence for reduction in the ppGpp degradation rate following an increase in pppGpp pool, during recovery from stringent response. From a genetic screen, the nudix hydrolases MutT and NudG were identified as over-expression suppressors of the growth defect of ΔspoT and ΔspoT ΔgppA strains. The effect of over-expression of these hydrolases on the stringent response to amino acid starvation and basal (p)ppGpp pool was studied. Over-expression of each hydrolase reduced the strength of the stringent response to amino acid starvation, and additionally, perturbed the ratio of ppGpp to pppGpp in strains with reduced SpoT hydrolase activity. In these strains that do not accumulate pppGpp during amino acid starvation, the expression of NudG or MutT supported pppGpp accumulation. This lends support to the idea that a reduction in the SpoT hydrolase activity is sufficient to cause the loss of pppGpp accumulation and therefore the phenomenon is independent of hydrolases that target pppGpp, such as GppA.

Plasticity, ligand conformation and enzyme action of Mycobacterium smegmatis MutT1.

Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.

Identification, Biosynthesis, and Decapping of NAD-Capped RNAs in B. subtilis.

The ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) decorates various RNAs in different organisms. In the proteobacterium Escherichia coli, the NAD-cap confers stability against RNA degradation. To date, NAD-RNAs have not been identified in any other bacterial microorganism. Here, we report the identification of NAD-RNA in the firmicute Bacillus subtilis. In the late exponential growth phase, predominantly mRNAs are NAD modified. NAD is incorporated de novo into RNA by the cellular RNA polymerase using non-canonical transcription initiation. The incorporation efficiency depends on the -1 position of the promoter but is independent of sigma factors or mutations in the rifampicin binding pocket. RNA pyrophosphohydrolase BsRppH is found to decap NAD-RNA. In vitro, the decapping activity is facilitated by manganese ions and single-stranded RNA 5' ends. Depletion of BsRppH influences the gene expression of ∼13% of transcripts in B. subtilis. The NAD-cap stabilizes RNA against 5'-to-3'-exonucleolytic decay by RNase J1.

Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.

Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.

DsRNA-mediated silencing of Nudix hydrolase in Trichinella spiralis inhibits the larval invasion and survival in mice.

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/µl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/µl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host.

Characterization and functional analysis of Trichinella spiralis Nudix hydrolase.

Trichinella spiralis Nudix hydrolase (TsNd) was identified by screening a T7 phage display cDNA library from T. spiralis intestinal infective larvae (IIL), and vaccination of mice with recombinant TsNd protein (rTsNd) or TsNd DNA vaccine produced a partial protective immunity. The aim of this study was to identify the characteristics and biological functions of TsNd in the process of invasion and development of T. spiralis larvae. Transcription and expression of TsNd gene at all developmental stages of T. spiralis were observed by qPCR and immunofluorescent test (IFT). The rTsNd had the Nd enzymatic activity to dGTP, NAD, NADP and CoA. Its kinetic properties on the preferred substrate dGTP were calculated, and the Vmax, Km, and kcat/Km values at pH 8.0 were 3.19 µM min(-1) µg(-1), 370 µM, and 144 s(-1) M(-1), respectively, in reaction matrix containing 5 mM Zn(2+) and 2 mM DTT. The rTsNd was active from 25 °C to 50 °C, with optimal activity at 37 °C. rTsNd was able to bind specifically to mouse intestinal epithelial cells (IECs) and promoted the larval invasion of IECs, whereas anti-rTsNd antibodies inhibited the larval invasion of IECs in a dose-dependent manner. Anti-rTsNd antibodies could kill T. spiralis infective larvae by an ADCC-mediated mechanism. Our results showed that the rTsNd protein was able to interact with host IECs, had the Nudix hydrolasing activity and the enzymatic activity appeared to be essential indispensable for the T. spiralis larval invasion, development and survival in host.