UMP-CMP kinase 2 inhibits ZIKV replication through activation of type I IFN signaling pathway.

Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the association and the underlying mechanisms between CMPK2 induction and ZIKV replication remain to be determined. We investigated the induction of CMPK2 during ZIKV infection and the effect of CMPK2 on ZIKV replication in A549, U251, Vero, IFNAR-deficient U5A and its parental 2fTGH cells, Huh7 and its RIG-I-deficient derivatives Huh7.5.1 cells. The activation status of Jak-STAT signaling pathway was determined by detecting the phosphorylation level of STAT1, the activity of interferon stimulated response element (ISRE) and the expression of several interferon stimulated genes (ISGs). We found that ZIKV infection induced CMPK2 expression through an IFNAR and RIG-I dependent manner. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication in A549 and U251 cells. Mechanically, we found that CMPK2 overexpression increased IFNß expression and activated Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the upregulated expression of downstream ISGs. These findings suggest that ZIKV infection induced CMPK2 expression, which inhibited ZIKV replication and serves as a positive feedback regulator for IFN-Jak/STAT pathway.

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

The selected genes NR6A1, RSAD2-CMPK2, and COL3A1 contribute to body size variation in Meishan pigs through different patterns.

The high-fertility Meishan pig is currently categorized into medium sized (MMS) and small sized (SMS) based on body size. To identify causal genes responsible for the variation in body size within the two categories, we sequenced individuals representing the entire consanguinity of the existing Meishan pig. This enabled us to conduct genome selective signal analysis. Our findings revealed the genomes of MMS and SMS are stratified, with selective sweep regions formed by differential genomic intervals between the two categories enriched in multiple pig body size related quantitative trait loci (QTLs). Furthermore, the missense mutation c.575T > C of candidate causal gene NR6A1, accounting for the variation in lumbar vertebrae number in pigs, was positively selected in MMS only, leading to an increase in body length of MMS at 6 months of age. To precisely identify causal genes accounting for body size variation through multi-omics, we collected femoral cartilage and liver transcription data from MMS and SMS respectively, and re-sequencing data from pig breeds exhibiting varying body sizes. We found that two selected regions where the RSAD2-CMPK2 and COL3A1 genes are located, respectively, showed different haplotypes in pig breeds of varying body size, and was associated with body or carcass length in hybridized Suhuai pig. Additionally, the above three hub genes, were significantly greater expressed in SMS femoral cartilage and liver tissues compared to MMS. These three genes could strengthen the pathways related to bone resorption and metabolism in SMS, potentially hindering bone and skeletal development and resulting in a smaller body size in SMS. These findings provide valuable insights into the genetic mechanism of body size variation in Meishan pig population.

The existing well-known Meishan pig population has been categorized into medium sized (MMS), and small sized (SMS) based on body size, which is a result of artificial selection. MMS is relatively large in all body size traits, but otherwise have highly similar appearance and performance traits. To effectively identify the candidate selected genes that contribute to the body size variation in Meishan pigs, this study collected individuals from all lineages of MMS and SMS for re-sequencing. Additionally, femoral cartilage and liver transcription data were collected from MMS and SMS, respectively, and re-sequencing data from pig breeds exhibiting varying body sizes were also analyzed. Through multi-omics analysis, it was discovered that the missense mutation c.575T > C in the candidate causal gene NR6A1 was positively selected in MMS only, leading to an increase in the body length of MMS at 6 months of age. Moreover, the selected genes RSAD2-CMPK2 and COL3A1 were found to be significantly greater expressed in SMS femoral cartilage and liver tissues compared with MMS. These genes could potentially strengthen bone resorption and metabolism-related pathways in SMS. These findings contribute to a better understanding of the genetic mechanisms underlying body size variation in Meishan pigs and Chinese indigenous pigs.

The evolving landscape of involvement of DTYMK enzymes in cancer.

Cancer cells require continuous synthesis of nucleotides for their uncontrolled proliferation. Deoxy thymidylate kinase (DTYMK) belongs to the thymidylate kinase family and is concerned with pyrimidine metabolism. DTYMK catalyzes the ATP-based conversion of deoxy-TMP to deoxy-TDP in both de novo and salvage pathways. Different studies demonstrated that DTYMK was increased in various types of cancers such as hepatocellular carcinoma, colon cancer, lung cancer, etc. Increased level of DTYMK was associated with poorer survival and prognosis, stage, grade and size of tumor, cell proliferation, colony formation, enhanced sensitivity to chemotherapy drugs, migration. Some studies were showed that knockdown of DTYMK reduced the signaling pathway of PI3K/AKT and downregulated expression of CART, MAPKAPK2, AKT1 and NRF1. Moreover, some microRNAs could suppress DTYMK expressions. On the other hand based on the TIMER database, the infiltration of macrophages, dendritic cells, neutrophils, B cells, CD4+ T cell and CD8+ T cell is affected by DTYMK. In the present review, we describe the genomic location, protein structure and isoforms of DTYMK and focus on its role in cancer development.

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases.

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T. maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T. maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Bone marrow mesenchymal stem cell-derived exosomes miR-202-5p inhibited pyroptosis to alleviate lung ischemic-reperfusion injury by targeting CMPK2.

Bone mesenchymal stem cell-derived exosome (BMSC-exosome) is a potential candidate for lung ischemia-reperfusion injury (LIRI) treatment. This study aims to investigate the anti-pyroptosis effect of BMSC-exosomes in LIRI. The LIRI cell model was established by hypoxia/reoxygenation (H/R) treatment. Interleukin (IL)-1ß and IL-18 levels were examined by enzyme-linked immunosorbent assay. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Lactate dehydrogenase (LDH) release was examined using a LDH assay kit. The interaction between microRNA (miR)-202-5p and cytidine monophosphate kinase 2 (CMPK2) was analyzed using dual-luciferase reporter assay and RNA immunoprecipitation. BMSC-exosomes promoted cell viability and suppressed pyroptosis in H/R-treated mouse lung epithelial. miR-202-5p was enriched in BMSC-exosomes, and exosomal miR-202-5p inhibition upregulated pyroptosis-associated proteins, including cleaved N-terminal Gasdermin D, nucleotide-binding domain-like receptor family member pyrin domain-containing protein 3, and Caspase1. Meanwhile, miR-202-5p suppressed CMPK2 expression by directly targeting CMPK2. Expectedly, CMPK2 knockdown reversed the promoting effect of exosomal miR-202-5p inhibition on pyroptosis in LIRI. Therefore, BMSC-derived exosome miR-202-5p repressed pyroptosis to inhibit LIRI progression by targeting CMPK2.

Identification of novel potential inhibitor of thymidylate kinase from Variola virus.

A hit compound was designed using Fragment Based Drug Designing (FBDD) approach, density functional theory (DFT) calculations were performed to find the structural and electronic properties. Additionally, pharmacokinetic properties were studied to understand the biological response of the compound. Docking studies were carried out with the protein structure of VrTMPK and HssTMPK with the reported hit compound. The favored docked complex was further carried to perform MD simulations; the RMSD plot and H-bond analysis was done for 200 ns. Also, MM-PBSA was done to understand the binding energy constituents and stability of the complex. A comparative study of the designed hit compound was done with FDA approved Tecovirimat. As a result, it was found that the reported compound (POX-A)is a potential selective inhibitor for Variola virus. Hence, it can be used to study further in vivo and in vitro behavior of the compound.Communicated by Ramaswamy H. Sarma.

Computational aided design of a halotolerant CMP kinase for enzymatic synthesis of cytidine triphosphate.

The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200 mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6 mM, and the productivity was 12.6 mM L-1 h-1, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.

Cell-free regeneration of ATP based on polyphosphate kinase 2 facilitates cytidine 5'-monophosphate production.

Cytidine 5'-monophosphate (5'-CMP), a key intermediate for the production of nucleotide derivatives, has been extensively used in food, agriculture, and medicine industries. Compared to RNA degradation and chemical synthesis, the biosynthesis of 5'-CMP has attracted wide attention due to its relatively low cost and eco-friendliness. In this study, we developed a cell-free regeneration of ATP based on polyphosphate kinase 2 (PPK2) to manufacture 5'-CMP from cytidine (CR). McPPK2 from Meiothermus cerbereus exhibited high specific activity (128.5 U/mg) and was used to accomplish ATP regeneration. McPPK2 and LhUCK (a uridine-cytidine kinase from Lactobacillus helveticus) were combined to convert CR to 5'-CMP. Further, the degradation of CR was inhibited by knocking out cdd from the Escherichia coli genome to enhance 5'-CMP production. Finally, the cell-free system based on ATP regeneration maximized the titer of 5'-CMP up to 143.5 mM. The wider applicability of this cell-free system was demonstrated in the synthesis of deoxycytidine 5'-monophosphate (5'-dCMP) from deoxycytidine (dCR) by incorporating McPPK2 and BsdCK (a deoxycytidine kinase from Bacillus subtilis). This study suggests that the cell-free regeneration of ATP based on PPK2 has the advantage of great flexibility for producing 5'-(d)CMP and other (deoxy)nucleotides.

New tetrahydropyrimidine-1,2,3-triazole clubbed compounds: Antitubercular activity and Thymidine Monophosphate Kinase (TMPKmt) inhibition.

Two series of new tetrahydropyrimidine (THPM)-1,2,3-triazole clubbed compounds were designed, synthesized and screened for their antitubercular (anti-TB) activity against M. tuberculosis H37Rv strain using microplate alamar blue assay (MABA). The most active compounds 5c, 5d, 5e and 5f were further examined for their cytotoxicity against the growth of RAW 264.7 mouse macrophage cells using MTT assay. The four compounds showed safety profiles better than or comparable to that of ethambutol (EMB). These compounds were evaluated for their inhibition activity against mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt). Compounds 5c and 5e were the most potent exhibiting comparable inhibition activity to that of the natural substrate deoxythymidine monophosphate (dTMP). An in silico study was performed including docking of the most active compounds 5c and 5e into the TMPKmt (PDB: ID 1G3U) binding pocket in addition to prediction of their physicochemical and pharmacokinetic properties to explore the overall activity of these anti-TB candidates. Compounds 5c and 5e are promising anti-TB agents and TMPKmt inhibitors with acceptable oral bioavailability, physicochemical and pharmacokinetic properties.

Targeting the Plasmodium falciparum's Thymidylate Monophosphate Kinase for the Identification of Novel Antimalarial Natural Compounds.

Recent reports of resistance to artemisinin-based combination drugs necessitate the need to discover novel antimalarial compounds. The present study was aimed at identifying novel antimalarial compounds from natural product libraries using computational methods. Plasmodium falciparum is highly dependent on the pyrimidine biosynthetic pathway, a de novo pathway responsible for the production of pyrimidines, and the parasite lacks the pyrimidine salvage enzymes. The P. falciparum thymidylate monophosphate kinase (PfTMPK) is an important protein necessary for rapid DNA replication; however, due to its broad substrate specificity, the protein is distinguished from its homologs, making it a suitable drug target. Compounds from AfroDB, a database of natural products originating from Africa, were screened virtually against PfTMPK after filtering the compounds for absorption, distribution, metabolism, excretion, and toxicity (ADMET)-acceptable compounds with FAF-Drugs4. Thirteen hits with lower binding energies than thymidine monophosphate were selected after docking. Among the thirteen compounds, ZINC13374323 and ZINC13365918 with binding energies of -9.4 and -8.9 kcal/mol, respectively, were selected as plausible lead compounds because they exhibited structural properties that ensure proper binding at the active site and inhibitory effect against PfTMPK. ZINC13374323 (also called aurantiamide acetate) is known to exhibit anti-inflammatory and antiviral activities, and ZINC13365918 exhibits antileishmanial activity. Furthermore, aurantiamide acetate, which is commercially available, is a constituent of Artemisia annua, the herb from which artemisinin was derived. The compound also shares interactions with several residues with a potent thymidine analog inhibitor of PfTMPK. The anti-plasmodial activity of aurantiamide acetate was evaluated in vitro, and the mean half-maximal inhibitory concentration (IC50) was 69.33 µM when synchronized P. falciparum 3D7 culture was used as compared to IC50 > 100 µM with asynchronized culture. The significance of our findings within the context of malaria treatment strategies and challenges is discussed.

Electroacupuncture Inhibits NLRP3 Activation by Regulating CMPK2 After Spinal Cord Injury.

Objectives: This study aimed to evaluate the expression of cytosine monophosphate kinase 2 (CMPK2) and activation of the NLRP3 inflammasome in rats with spinal cord injury (SCI) and to characterize the effects of electroacupuncture on CMPK2-associated regulation of the NLRP3 inflammasome. Methods: An SCI model was established in Sprague-Dawley (SD) rats. The expression levels of NLRP3 and CMPK2 were measured at different time points following induction of SCI. The rats were randomly divided into a sham group (Sham), a model group (Model), an electroacupuncture group (EA), an adeno-associated virus (AAV) CMPK2 group, and an AAV NC group. Electroacupuncture was performed at jiaji points on both sides of T9 and T11 for 20 min each day for 3 consecutive days. In the AAV CMPK2 and AAV NC groups, the viruses were injected into the T9 spinal cord via a microneedle using a microscope and a stereotactic syringe. The Basso-Beattie-Bresnahan (BBB) score was used to evaluate the motor function of rats in each group. Histopathological changes in spinal cord tissue were detected using H&E staining, and the expression levels of NLRP3, CMPK2, ASC, caspase-1, IL-18, and IL-1ß were quantified using Western blotting (WB), immunofluorescence (IF), and RT-PCR. Results: The expression levels of NLRP3 and CMPK2 in the spinal cords of the model group were significantly increased at day 1 compared with those in the sham group (p < 0.05). The expression levels of NLRP3 and CMPK2 decreased gradually over time and remained low at 14 days post-SCI. We successfully constructed AAV CMPK2 and showed that CMPK2 was significantly knocked down following 2 dilutions. Finally, treatment with EA or AAV CMPK2 resulted in significantly increased BBB scores compared to those in the model group and the AAV NC group (p < 0.05). The histomorphology of the spinal cord in the EA and AAV CMPK2 groups was significantly different than that in the model and AAV NC groups. WB, IF, and PCR analyses showed that the expression levels of CMPK2, NLRP3, ASC, caspase-1, IL-18, and IL-1ß were significantly lower in the EA and AAV CMPK2 groups compared with those in the model and AAV NC groups (p < 0.05). Conclusion: Our study showed that CMPK2 regulated NLRP3 expression in rats with SCI. Activation of NLRP3 is a critical mechanism of inflammasome activation and the inflammatory response following SCI. Electroacupuncture downregulated the expression of CMPK2 and inhibited activation of NLRP3, which could improve motor function in rats with SCI.

Heavy metal tolerance of Mesorhizobium delmotii thymidylate kinase.

Metal ions play an important role in many metabolic processes in all living organisms. At low concentrations, heavy metals such as Fe2+, Cu2+ and Zn2+ are essential cofactors for many enzymes. However, at high concentrations they are toxic. Mesorhizobium species belong to the class α-proteobacteria and have high tolerance to soil acidity, salinity, temperature extremes, and metallicolous conditions. To identify factors responsible for this tolerance we have studied the effects of metal ions on Mesorhizobium delmotii thymidylate kinase (MdTMPK), an essential enzyme in the synthesis of dTTP, thus being vital for cell growth. We show that Mg2+ and Mn2+ are the divalent metal ions required for catalysis and that Mn2+ gives the highest catalytic efficiency. MdTMPK activity in the presence of Mg2+ was strongly inhibited by the co-presence of Zn2+, Ni2+ and Co2+. However, the addition of Cs+ caused >2-fold enhanced MdTMPK activity. For TMPK from Bacilus anthracis and humans, the effects of Mg2+ and Mn2+ were similar, whereas the effects of other divalent metal ions were different, and no stimulatory effect of Cs+ was observed. Together, our results demonstrate that MdTMPK and BaTMPK function well in the presence of high concentrations of heavy metal ions, introducing a potential contribution of these enzymes to the heavy metal tolerance of Mesorhizobium delmotii and Bacillus anthracis.

Differential expression of enzymes in thymidylate biosynthesis in zebrafish at different developmental stages: implications for dtymk mutation-caused neurodegenerative disorders.

BACKGROUND: Deoxythymidine triphosphate (dTTP) is an essential building block of DNA, and defects in enzymes involved in dTTP synthesis cause neurodegenerative disorders. For instance, mutations in DTYMK, the gene coding for thymidylate kinase (TMPK), cause severe microcephaly in human. However, the mechanism behind this is not well-understood. Here we used the zebrafish model and studied (i) TMPK, an enzyme required for both the de novo and the salvage pathways of dTTP synthesis, and (ii) thymidine kinases (TK) of the salvage pathway in order to understand their role in neuropathology. RESULTS: Our findings reveal that maternal-stored dNTPs are only sufficient for 6 cell division cycles, and the levels of dNTPs are inversely correlated to cell cycle length during early embryogenesis. TMPK and TK activities are prominent in the cytosol of embryos, larvae and adult fish and brain contains the highest TMPK activity. During early development, TMPK activity increased gradually from 6 hpf and a profound increase was observed at 72 hpf, and TMPK activity reached its maximal level at 96 hpf, and remained at high level until 144 hpf. The expression of dtymk encoded Dtymk protein correlated to its mRNA expression and neuronal development but not to the TMPK activity detected. However, despite the high TMPK activity detected at later stages of development, the Dtymk protein was undetectable. Furthermore, the TMPK enzyme detected at later stages showed similar biochemical properties as the Dtymk enzyme but was not recognized by the Dtymk specific antibody. CONCLUSIONS: Our results suggest that active dNTP synthesis in early embryogenesis is vital and that Dtymk is essential for neurodevelopment, which is supported by a recent study of dtymk knockout zebrafish with neurological disorder and lethal outcomes. Furthermore, there is a novel TMPK-like enzyme expressed at later stages of development.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Structural and functional analysis of human thymidylate kinase isoforms.

Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK, i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.

DTYMK is essential for genome integrity and neuronal survival.

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Inhibition of DTYMK significantly restrains the growth of HCC and increases sensitivity to oxaliplatin.

Most patients with hepatocellular carcinoma (HCC) are in the middle or advanced stage at the time of diagnosis, and the therapeutic effect is limited. Therefore, this study aimed to verify whether deoxythymidylate kinase (DTYMK) increased in HCC and was an effective therapeutic target in HCC. The findings revealed that the DTYMK level significantly increased and correlated with poor prognosis in HCC. However, nothing else is known, except that DTYMK could catalyze the phosphorylation of deoxythymidine monophosphate (dTMP) to form deoxythymidine diphosphate (dTDP). A number of experiments were performed to study the function of DTYMK in vitro and in vivo to resolve this knowledge gap. The knockdown of DTYMK was found to significantly inhibit the growth of HCC and increase the sensitivity to oxaliplatin, which is commonly used in HCC treatment. Moreover, DTYMK was found to competitively combine with miR-378a-3p to maintain the expression of MAPK activated protein kinase 2 (MAPKAPK2) and thus activate the phospho-heat shock protein 27 (phospho-HSP27)/nuclear factor NF-kappaB (NF-κB) axis, which mediated the drug resistance, proliferation of tumor cells, and infiltration of tumor-associated macrophages by inducing the expression of C-C motif chemokine ligand 5 (CCL5). Thus, this study demonstrated a new mechanism and provided a new insight into the role of mRNA in not only encoding proteins to regulate the process of life but also regulating the expression of other genes and tumor microenvironment through the competing endogenous RNA (ceRNA) mechanism.

Multifaceted impact of a nucleoside monophosphate kinase on 5'-end-dependent mRNA degradation in bacteria.

A key pathway for mRNA degradation in bacterial cells begins with conversion of the initial 5'-terminal triphosphate to a monophosphate, a modification that renders transcripts more vulnerable to attack by ribonucleases whose affinity for monophosphorylated 5' ends potentiates their catalytic efficacy. In Escherichia coli, the only proteins known to be important for controlling degradation via this pathway are the RNA pyrophosphohydrolase RppH, its heteromeric partner DapF, and the 5'-monophosphate-assisted endonucleases RNase E and RNase G. We have now identified the metabolic enzyme cytidylate kinase as another protein that affects rates of 5'-end-dependent mRNA degradation in E. coli. It does so by utilizing two distinct mechanisms to influence the 5'-terminal phosphorylation state of RNA, each dependent on the catalytic activity of cytidylate kinase and not its mere presence in cells. First, this enzyme acts in conjunction with DapF to stimulate the conversion of 5' triphosphates to monophosphates by RppH. In addition, it suppresses the direct synthesis of monophosphorylated transcripts that begin with cytidine by reducing the cellular concentration of cytidine monophosphate, thereby disfavoring the 5'-terminal incorporation of this nucleotide by RNA polymerase during transcription initiation. Together, these findings suggest dual signaling pathways by which nucleotide metabolism can impact mRNA degradation in bacteria.