Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

Uridine-cytidine kinase 2 potentiates the mutagenic influence of the antiviral ß-d-N4-hydroxycytidine.

Molnupiravir (EIDD-2801) is an antiviral that received approval for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. Treatment of bacteria or cell lines with the active form of molnupiravir, ß-d-N4-hydroxycytidine (NHC, or EIDD-1931), induces mutations in DNA. Yet these results contrast in vivo genotoxicity studies conducted during registration of the drug. Using a CRISPR screen, we found that inactivating the pyrimidine salvage pathway component uridine-cytidine kinase 2 (Uck2) renders cells more tolerant of NHC. Short-term exposure to NHC increased the mutation rate in a mouse myeloid cell line, with most mutations being T:A to C:G transitions. Inactivating Uck2 impaired the mutagenic activity of NHC, whereas over-expression of Uck2 enhanced mutagenesis. UCK2 is upregulated in many cancers and cell lines. Our results suggest differences in ribonucleoside metabolism contribute to the variable mutagenicity of NHC observed in cancer cell lines and primary tissues.

Characterization of uridine-cytidine kinase like-1 nucleoside kinase activity and its role in tumor growth.

Uridine-cytidine kinase like-1 (UCKL-1) is a largely uncharacterized protein with high sequence similarity to other uridine-cytidine kinases (UCKs). UCKs play an important role in the pyrimidine salvage pathway, catalyzing the phosphorylation of uridine and cytidine to UMP and CMP, respectively. Only two human UCKs have been identified, UCK1 and UCK2. Previous studies have shown both enzymes phosphorylate uridine and cytidine using ATP as the phosphate donor. No studies have evaluated the kinase potential of UCKL-1. We cloned and purified UCKL-1 and found that it successfully phosphorylated uridine and cytidine using ATP as the phosphate donor. The catalytic efficiency (calculated as kcat/KM) was 1.2 × 104 s-1, M-1 for uridine and 0.7 × 104 s-1, M-1 for cytidine. Our lab has previously shown that UCKL-1 is up-regulated in tumor cells, providing protection against natural killer (NK) cell killing activity. We utilized small interfering RNA (siRNA) to down-regulate UCKL-1 in vitro and in vivo to determine the effect of UCKL-1 on tumor growth and metastasis. The down-regulation of UCKL-1 in YAC-1 lymphoma cells in vitro resulted in decreased cell counts and increased apoptotic activity. Down-regulation of UCKL-1 in K562 leukemia cells in vivo led to decreased primary tumor growth and less tumor cell dissemination and metastasis. These results identify UCKL-1 as a bona fide pyrimidine kinase with the therapeutic potential to be a target for tumor growth inhibition and for diminishing or preventing metastasis.

Targeting uridine-cytidine kinase 2 induced cell cycle arrest through dual mechanism and could improve the immune response of hepatocellular carcinoma.

BACKGROUND: Pyrimidine metabolism is critical for tumour progression. Uridine-cytidine kinase 2 (UCK2), a key regulator of pyrimidine metabolism, is elevated during hepatocellular carcinoma (HCC) development and exhibits carcinogenic effects. However, the key mechanism of UCK2 promoting HCC and the therapeutic value of UCK2 are still undefined. The aim of this study is to investigate the potential of UCK2 as a therapeutic target for HCC. METHODS: Gene expression matrices were obtained from public databases. RNA-seq, co-immunoprecipitation and RNA-binding protein immunoprecipitation were used to determine the mechanism of UCK2 promoting HCC. Immune cell infiltration level and immune-related functional scores were evaluated to assess the link between tumour microenvironment and UCK2. RESULTS: In HCC, the expression of UCK2 was upregulated in part by TGFß1 stimulation. UCK2 promoted cell cycle progression of HCC by preventing the degradation of mTOR protein and maintaining the stability of PDPK1 mRNA. We also identified UCK2 as a novel RNA-binding protein. Downregulation of UCK2 induced cell cycle arrest and activated the TNFα/NFκB signalling pathway-related senescence-associated secretory phenotype to modify the tumour microenvironment. Additionally, UCK2 was a biomarker of the immunosuppressive microenvironment. Downregulated UCK2 induced a secretory phenotype, which could improve the microenvironment, and decreased UCK2 remodelling metabolism could lower the resistance of tumour cells to T-cell-mediated killing. CONCLUSIONS: Targeting UCK2 inhibits HCC progression and could improve the response to immunotherapy in patients with HCC. Our study suggests that UCK2 could be an ideal target for HCC.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. METHODS: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. RESULTS: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. CONCLUSIONS: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Identification of a four-gene metabolic signature predicting overall survival for hepatocellular carcinoma.

While hundreds of consistently altered metabolic genes had been identified in hepatocellular carcinoma (HCC), the prognostic role of them remains to be further elucidated. Messenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and GSE14520 data set from the Gene Expression Omnibus database. Univariate Cox regression analysis and lasso Cox regression model established a novel four-gene metabolic signature (including acetyl-CoA acetyltransferase 1, glutamic-oxaloacetic transaminase 2, phosphatidylserine synthase 2, and uridine-cytidine kinase 2) for HCC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was significantly correlated with other negative prognostic factors such as higher α-fetoprotein. The signature was found to be an independent prognostic factor for HCC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. Furthermore, gene set enrichment analyses revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust four-gene metabolic signature for HCC prognosis prediction. The signature might reflect the dysregulated metabolic microenvironment and provided potential biomarkers for metabolic therapy and treatment response prediction in HCC.

Uridine-cytidine kinase 2 (UCK2): A potential diagnostic and prognostic biomarker for lung cancer.

Lung cancer has the highest morbidity and mortality among all cancers. Discovery of early diagnostic and prognostic biomarkers of lung cancer can greatly facilitate the survival rate and reduce its mortality. In our study, by analyzing Gene Expression Omnibus and Oncomine databases, we found a novel potential oncogene uridine-cytidine kinase 2 (UCK2), which was overexpressed in lung tumor tissues compared to adjacent nontumor tissues or normal lung. Then we confirmed this finding in clinical samples. Specifically, UCK2 was identified as highly expressed in stage IA lung cancer with a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.9). We also found that high UCK2 expression was related to poorer clinicopathological features, such as higher T stage and N stage and higher probability of early recurrence. Furthermore, we found that patients with high UCK2 expression had poorer first progression survival and overall survival than patients with low UCK2 expression. Univariate and multivariate Cox regression analyses showed that UCK2 was an independent risk factor related with worse DFS and OS. By gene set enrichment analysis, tumor-associated biological processes and signaling pathways were enriched in the UCK2 overexpression group, which indicated that UCK2 might play a vital role in lung cancer. Furthermore, in cytology experiments, we found that knockdown of UCK2 could suppress the proliferation and migration of lung cancer cells. In conclusion, our study indicated that UCK2 might be a potential early diagnostic and prognostic biomarker for lung cancer.

Uridine-cytidine kinase 2 upregulation predicts poor prognosis of hepatocellular carcinoma and is associated with cancer aggressiveness.

Patients with advanced hepatocellular carcinoma (HCC) continue to have a dismal prognosis. Potential biomarkers to determine prognosis and select targeted therapies are urgently needed for patients with HCC. This study aimed to elucidate the role of UCK2 in HCC prognosis and tumor progression. We performed a screen of public databases to identify functional genes associated with HCC tumorigenesis, progression, and outcome. We identified uridine-cytidine kinase 2 (UCK2) as a gene of interest for further study. UCK2 promoting HCC aggressiveness was demonstrated by evaluation of clinical samples, in vitro experiments, in vivo tumorigenicity, and transcript analysis. UCK2 expression was generally elevated in HCC and was significantly correlated with poor survival and inferior clinicopathological characteristics of HCC patients. A multivariate analysis revealed that high UCK2 expression was an independent factor for poor prognosis. In HCC cell lines, UCK2 knockdown suppressed cell migration and invasion and inhibited cell proliferation, while UCK2 overexpression had an opposite effect. Animal model experiments confirmed that knockdown of UCK2 suppressed tumor growth in vivo. The bioinformatics analysis demonstrated that UCK2 might associated with metabolsim, splicesome, and adherens junction. UCK2 is highly associated with HCC malignant behavior and is a potential prognostic predictor for HCC patients in the clinic.

UCK2 upregulation might serve as an indicator of unfavorable prognosis of hepatocellular carcinoma.

Uridine-cytidine kinases (encoded by UCK1, UCKL1, and UCK2) catalyze the phosphorylation of uridine and cytidine to uridine monophosphate (UMP) and cytidine monophosphate (CMP). In this study, using data from the Cancer Genome Atlas (TCGA), we analyzed the expression profile of uridine-cytidine kinase genes in hepatocellular carcinoma (HCC), their prognostic value, and the epigenetic alterations associated with their dysregulation. Results showed that UCKL1 and UCK2, but not UCK1 were significantly upregulated in HCC tissues than in adjacent normal tissues. Only UCK2 was significantly upregulated in the deceased group and the recurrence group, compared to the control groups. Multivariate analysis confirmed that increased UCK2 expression was an independent prognostic indicator of shorter overall survival (OS) (HR: 1.760, 95% CI: 1.398-2.216, P < 0.001) and recurrence-free survival (RFS) (HR: 1.543, 95% CI: 1.232-1.933, P < 0.001). Two CpG sites (cg09277749 and cg21143899) were significantly hypomethylated in HCC tissues than in adjacent normal tissues and were negatively correlated with UCK2 expression. However, survival analysis showed that only high methylation of cg0927774 was associated with better OS and RFS of HCC patients. Based on the findings above, we infer that UCK2 upregulation might be a valuable prognostic marker in HCC. The methylation of status cg0927774 might play a critical role in its expression. © 2018 IUBMB Life, 71(1):105-112, 2019.

Impact of the Uridine⁻Cytidine Kinase Like-1 Protein and IL28B rs12979860 and rs8099917 SNPs on the Development of Hepatocellular Carcinoma in Cirrhotic Chronic Hepatitis C Patients-A Pilot Study.

Background and objectives: The hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma (HCC) in the western world. The efficacy of surveillance programs for early detection of HCC is not satisfactory: many tumors are diagnosed at the late, incurable stages. Therefore, there is a need in reliable prognostic markers for the proper follow-up of HCV-positive patients. The aim of the present study was to assess the prognostic value of the uridine⁻cytidine kinase-like protein 1 (UCKL-1), a putative oncoprotein, together with genetically determined polymorphisms in the interleukin 28B (IL28B) gene (rs12979860, rs8099917) in the development of HCC in HCV-positive cirrhotic patients. Materials and Methods: We included 32 HCV cirrhotic patients, 21 (65.6%) of whom had HCC. The expression of UCKL-1 was assessed in liver tissue sections, using immunohistochemistry. For IL28B rs12979860 and rs8099917 genotype analysis, the corresponding genomic regions were amplified by polymerase chain reaction (PCR) with appropriate primers. Results: We have found that UCKL-1 expression was significantly increased in HCC (p = 0.003). The presence of rs8099917 TT single-nucleotide polymorphism (SNP) elevated the chances of HCC manifestation more than sevenfold (OR = 7.3, p = 0.0273). The presence of rs12979860 CC SNP also heightened HCC chances more than sevenfold (OR = 7.5, p = 0.0765). Moreover, in the HCC group, a combination of IL28B rs12979860 non-TT and rs8099917 TT genotypes was observed more often, compared with the non-HCC group. Other combinations of IL28B rs12979860 and rs8099917 SNIPs were associated with a reduced risk of HCC development, approximately at the same extent. Conclusions: The presence of IL28B rs8099917 TT and rs12979860 CC SNPs, but not the intensity of UCKL-1 expression, is strongly associated with increased chances of HCC development in HCV-positive cirrhotic patients.

The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Testicular germ cell tumor susceptibility associated with the UCK2 locus on chromosome 1q23.

Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case-control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined = 6.0 × 10(-9)). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed (PFBAT = 2.3 × 10(-3)). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.

Plastid uridine salvage activity is required for photoassimilate allocation and partitioning in Arabidopsis.

Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning.

Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage.

Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.

Ehrlichia chaffeensis replication sites in adult Drosophila melanogaster.

Ehrlichia chaffeensis is a Gram-negative, obligate intracellular bacterium which causes the tick-borne disease human monocytic ehrlichiosis. In vertebrates, E. chaffeensis replicates in monocytes and macrophages. However, no clear cell or tissue tropism has been defined in arthropods. Our group identified two host genes that control E. chaffeensis replication and infection in vivo in Drosophila, Uridine cytidine kinase and separation anxiety. Using the UAS-GAL4 RNAi system, we generated F1 flies (UAS-gene of interestRNAi x tissue-GAL4 flies) that have Uck2 or san silenced in ubiquitous or tissue-specific fashion. When Uck2 or san were suppressed in the hemocytes or in the fat body, E. chaffeensis replicated poorly and caused significantly less severe infections. Silencing of these genes in the eyes, wings, or the salivary glands did not impact fly susceptibility or bacterial replication. Our data suggest that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals.

Identification of critical host mitochondrion-associated genes during Ehrlichia chaffeensis infections.

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis (HME). To determine what host components are important for bacterial replication, we performed microarray analysis on Drosophila melanogaster S2 cells by comparing host gene transcript levels between permissive and nonpermissive conditions for E. chaffeensis growth. Five-hundred twenty-seven genes had increased transcript levels unique to permissive growth conditions 24 h postinfection. We screened adult flies that were mutants for several of the "permissive" genes for the ability to support Ehrlichia replication. Three additional D. melanogaster fly lines with putative mutations in pyrimidine metabolism were also tested. Ten fly lines carrying mutations in the genes CG6479, separation anxiety, chitinase 11, CG6364 (Uck2), CG6543 (Echs1), withered (whd), CG15881 (Ccdc58), CG14806 (Apop1), CG11875 (Nup37), and dumpy (dp) had increased resistance to infection with Ehrlichia. Analysis of RNA by quantitative real-time reverse transcription-PCR (qRT-PCR) confirmed that the bacterial load was decreased in these mutant flies compared to wild-type infected control flies. Seven of these genes (san, Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) encoded proteins that had mitochondrial functions or could be associated with proteins with mitochondrial functions. Treatment of THP-1 cells with double-stranded RNA to silence the human UCK2 gene indicates that the disruption of the uridine-cytidine kinase affects E. chaffeensis replication in human macrophages. Experiments with cyclopentenyl cytosine (CPEC), a CTP synthetase inhibitor and cytosine, suggest that the nucleotide salvage pathway is essential for E. chaffeensis replication and that it may be important for the provision of CTP, uridine, and cytidine nucleotides.

A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine.

The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.

Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network.

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

Efficient One-Pot Synthesis of Cytidine 5'-Monophosphate Using an Extremophilic Enzyme Cascade System.

A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5'-monophosphate (5'-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5'-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5'-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5'-CMP. After optimization, 96 mM 5'-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5'-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5'-CMP.