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Pre-eclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation. It seriously affects the health of maternity and the fetus. However, the pathogenesis of PE is still unknown. Decidualization deficiency is considered a contributing factor to the development of PE. CTP synthetase (CTPS) which is the rate-limiting enzyme in the CTP de novo biosynthesis, is essential for nucleic acid synthesis and cellular energy metabolism, and often appears as cytoophidium in many cell types. Here, we found that the expression of CTPS was significantly downregulated in decidual tissues of patients with severe PE compared with healthy pregnant women. During in vitro decidualization, changes in CTPS were accompanied by opposite fluctuation of the AMPK signaling pathway. Moreover, the downregulation of CTPS by glutamine analogs or CTPS small interfering RNA inhibited the decidualization process and the AMPK signaling pathway. Investigating the underlying mechanism of action by co-immunoprecipitation coupled with mass spectrometry showed that CTPS interacted with ATP synthase (ATPS) and maintained the content of ATP on Day 3 of decidualization. However, when combined with mitochondrial stress protein STRESS-70 instead of ATPS, the concentration of ATP on Day 6 of induction was reduced. Corresponding to this, CTPS was mainly distributes in the cytoplasm on Day 3 of induction, while it appeared both in the cytoplasm and the nucleus on Day 6 in decidualized cells, which was similar to that in cells before induction. In summary, we believe that CTPS plays an important role in decidualization by participating in energy metabolism. Abnormal expression of CTPS in decidualization would lead to abnormal decidualization and consequently result in the occurrence of PE.

Drug-resistant tuberculosis (TB) infections are on the rise and anti-tuberculosis drugs that inhibit Mycobacterium tuberculosis (M. tuberculosis) through a new novel mechanism could be an important component of evolving TB therapy. Pantothenate kinase (PanK) and CTP synthetase (PyrG) are both essential for de novo pyrimidine biosynthesis. Given the extensive knowledge base on de novo pyrimidine biosynthesis inhibition of M. tuberculosis growth and survival, these enzymes present an interesting opportunity for anti-mycobacterial drug discovery. A recent experimental study shows that CDD-823953 and GSK-735826A act as dual PanK and PyrG inhibitors, respectively. However, the molecular mechanisms of their selective inhibition remain elusive. Herein, density functional theory (DFT) calculation was applied to unveil the molecular and reactivity properties of two lead compounds targeting these enzymes in a shot. Molecular dynamics simulations were then employed to investigate the inhibitory mechanism as well as selectivity impact of these potential inhibitors for their enzymes. Computational modeling of the ligands and the enzyme–ligand systems reveal that CDD-823953 and GSK-735826A lead compounds can potentially inhibit both PanK and PyrG thereby creating a pathway via the use of double target approach in tuberculosis treatment.

SUMMARYThe newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.

3-Deazauridine (3DU) is a nucleoside analogue that was investigated for its antineoplastic activity in cancer patients. 3DU after its phosphorylation in cells to its 5’-triphosphate is a potent inhibitor of CTP synthetase. Inhibition of this enzyme results in a reduction in cellular CTP and inhibition of RNA synthesis. This action of 3DU can also inhibit RNA synthesis since the viral RNA polymerase requires CTP for replication of RNA. 3DU was reported to inhibit the in vitro replication of the RNA influenza virus at concentrations in the range of 40 µM (1) and should also have the potential to inhibit the replication of SARS-CoV-2. In a pilot clinical study, we treated a patient with acute lymphoid leukemia with 3DU at doses ranging from 120 to 360 mg/kg using a continuous intravenous infusion ranging from 40 to 72 hr. (2). The objective of the study was to determine if 3DU could enhance the antileukemic action of the inhibitor of DNA methylation, decitabine, and overcome the problem of drug resistance to this deoxycytidine analogue. Partial responses were observed indicating that pharmacological action of 3DU could accomplish this objective. There were minimal signs of toxicity in this clinical study on 3DU which exhibited plasma concentrations of this analogue in the range that inhibit RNA virus replication. This observation suggests that it would be safe to use 3DU in patients with COVID-19. 3DU has a rapid onset of action with a significant sign of antineoplastic action observable after a 4 h in vitro treatment of human leukemic cells. This pharmacological property rapid action of 3DU may be very important in patients with advanced COVID-19 to inhibit the virus is during its rapid replication phase and prevent progression of the disease to acute lung inflammation. It would be interesting to test this hypothesis in advanced COVID-19 patients at high risk for disease progression. For an initial pilot study I recommend a 36 h infusion of 3DU at a total dose of 120 mg/kg. It is possible that therapy with 3DU may have the potential to rescue patients with advance COVID-19. References 1. Shannon, W.M., Arnett, G., Schabel, F.M., Jr., 1972. 3-Deazauridine: inhibition of ribonucleic acid virus-induced cytopathogenic effects in vitro. Antimicrob. Agents Chemother. 2, 159–163. 2. Raynal, N.J., Momparler, L.F., Rivard, G.E., Momparler, R.L., 2011. 3-Deazauridine enhances the antileukemic action of 5-aza-2'-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase. Leuk. Res. 35, 10-118. Citation Format: Richard Momparler. Anticancer agent 3-deazauridine has the potential to be effective against COVID-19 due to its inhibition of replication of RNA viruses [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P06.

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase ( ligA ) and CTP synthetase ( pyrG ) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti- S. enterica antibody response.

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P‐ and K‐distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health‐promoting probiotic bacteria.

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic a...

The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1‐Å‐resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure‐based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.

© 2016 American Chemical Society. CTP synthetases catalyze the last step of pyrimidine biosynthesis and provide the sole de novo source of cytosine-containing nucleotides. As a central regulatory hub, they are regulated by ribonucleotide and enzyme concentration through ATP and UTP substrate availability, CTP product inhibition, GTP allosteric modification, and quaternary structural changes including the formation of CTP-inhibited linear polymers (filaments). Here, we demonstrate that nicotinamide redox cofactors are moderate inhibitors of Escherichia coli CTP synthetase (EcCTPS). NADH and NADPH are the most potent, and the primary inhibitory determinant is the reduced nicotinamide ring. Although nicotinamide inhibition is noncompetitive with substrates, it apparently enhances CTP product feedback inhibition and GTP allosteric regulation. Further, CTP and GTP also enhance each other's effects, consistent with the idea that NADH, CTP, and GTP interact with a common intermediate enzyme state. A filament-blocking mutation that reduces CTP inhibitory effects also reduced inhibition by GTP but not NADH. Protein-concentration effects on GTP inhibition suggest that, like CTP, GTP preferentially binds to the filament. All three compounds display nearly linear dose-dependent inhibition, indicating a complex pattern of cooperative interactions between binding sites. The apparent synergy between inhibitors, in consideration with physiological nucleotide concentrations, points to metabolically relevant inhibition by nicotinamides, and implicates cellular redox state as a regulator of pyrimidine biosynthesis.

In eukaryotes, only a fraction of replication origins fire at each S phase. Local histone acetylation was proposed to control firing efficiency of origins, but conflicting results were obtained. We report that local histone acetylation does not reflect origin efficiencies along the adenosine monophosphate deaminase 2 locus in mammalian fibroblasts. Reciprocally, modulation of origin efficiency does not affect acetylation. However, treatment with a deacetylase inhibitor changes the initiation pattern. We demonstrate that this treatment alters pyrimidine biosynthesis and decreases fork speed, which recruits latent origins. Our findings reconcile results that seemed inconsistent and reveal an unsuspected effect of deacetylase inhibitors on replication dynamics.

Prs [PRPP (phosphoribosyl pyrophosphate) synthetase] catalyses the transfer of pyrophosphate from ATP to ribose 5-phosphate, thereby activating the pentose sugar for incorporation into purine and pyrimidine nucleotides. The Saccharomyces cerevisiae genome contains five genes, PRS1–PRS5, whose products display characteristic PRPP and bivalent-cation-binding sites of Prs polypeptides. Deletion of one or more of the five PRS genes has far-reaching and unexpected consequences, e.g. impaired cell integrity, temperature-sensitivity and sensitivity to VPA (valproic acid) and LiCl. CTP pools in prs1Δ and prs3Δ are reduced to 12 and 31% of the wild-type respectively, resulting in an imbalance in phospholipid metabolism which may have an impact on the intracellular inositol pool which is affected by the administration of either VPA or LiCl. Overexpression of CTP synthetase in prs1Δ prs3Δ strains partially reverses the VPA-sensitive phenotype. Yeast two-hybrid screening revealed that Prs3 and the yeast orthologue of GSK3 (glycogen synthase kinase 3), Rim11, a serine/threonine kinase involved in several signalling pathways, interact with each other. Furthermore, Prs5, an essential partner of Prs3, which also interacts with GSK3 contains three neighbouring phosphorylation sites, typical of GSK3 activation. These studies on yeast PRPP synthetases bring together and expand the current theories for the mood-stabilizing effects of VPA and LiCl in bipolar disorder.

S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser(462) and Thr(455) were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.

Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target., Graphical Abstract, Highlights • Two compounds activated by EthA kill M. tuberculosis through PyrG inhibition • EthA metabolite is active against PyrG and M. tuberculosis growth • Definition of the mechanism of activation and validation of PyrG as a new drug target, CTP synthetase PyrG, essential in Mycobacterium tuberculosis, could represent a new potential drug target. With a multidisciplinary approach, Mori et al. identify two compounds killing growing and dormant mycobacteria through PyrG inhibition, and define their mechanism of action.

The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazollum bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [H-3]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED50) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED50 with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [H-3]dFdC-anabolism increased 6-44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro

In Bacillus subtilis and other Gram-positive bacteria, pyrimidine-mediated regulation of the pyrG gene, which encodes CTP synthetase, occurs through an attenuation mechanism involving an intrinsic transcription terminator in the pyrG leader region. Low intracellular levels of CTP prevent termination at the attenuator by a mechanism that requires the nontemplate strand sequence GGGC at the pyrG transcription initiation site (first G =+1) and the leader transcript sequence GCUCCC located at the 5′ end of the terminator RNA hairpin. In this study, we demonstrate that reiterative transcription adds G residues (up to at least 10) to the 5′ end of pyrG transcripts when B . subtilis cells are starved for pyrimidines but not when cells are grown with excess cytidine. Regulated repetitive addition of G residues, as well as pyrimidine-mediated pyrG regulation, requires the sequence GGGC or GGGT at the start of pyrG transcription. Mutational insertion of four extra G residues at the 5′ end of the pyrG transcript (i.e., 5′-GGGGGGGC) results in constitutive pyrG expression. We propose that the incorporation of extra G residues by reiterative transcription at the wild-type promoter occurs when normal transcription elongation is stalled at position +4 by low levels of the incoming substrate, CTP, during pyrimidine limitation. The poly(G) extensions on the 5′ ends of pyrG transcripts act to prevent transcription attenuation by base pairing with the sequence CUCCCUUUC located in the 5′ strand of the terminator hairpin. This control mechanism is likely to operate in other Gram-positive bacteria containing similar pyrG leader sequences.

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser36, Ser330, Ser354, and Ser454, contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser --Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the Vmax of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the Vmax of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser36, Ser330, Ser354, and Ser454 residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.

The de novo biosynthesis of pyrimidine nucleotides provides essential precursors for multiple growth-related events in higher eukaryotes. Assembled from ATP, bicarbonate and glutamine, the uracil and cytosine nucleotides are fuel for the synthesis of RNA, DNA, phospholipids, UDP sugars and glycogen. Over the past 2 decades considerable progress has been made in elucidating the mechanisms by which cellular pyrimidines are modulated to meet the needs of the cell. Recent studies demonstrate that CAD, a rate-limiting enzyme in the de novo synthesis of pyrimidines, is regulated through reversible phosphorylation, Myc-dependent transcriptional changes and caspase-mediated degradation. These studies point to increasing evidence for cooperation between key cell signaling pathways and basic elements of cellular metabolism, and suggest that these events have the potential to determine distinct cellular fates, including growth, differentiation and death. This review highlights some of the recent advances in the regulation of pyrimidine synthesis by growth-factor-stimulated signaling pathways.

CPEC is a potent inhibitor of CTP synthetase and causes depletion of CTP and dCTP pools. AraC is an analog of dCyd and a chemotherapeutic agent. Here, we demonstrate that, upon incubation with CPEC, both the anabolism and cytostatic effect of AraC in SK-N-BE(2)c neuroblastoma cells were increased. Cotreatment of CPEC (50-250 nM) and AraC (37.5-500 nM) decreased the 4-day ED(50) value for AraC 2- to 8-fold in the SK-N-BE(2)c cell line, while pretreatment with CPEC followed by incubation with AraC alone decreased the 4-day ED(50) value for AraC 1- to 19-fold. Preincubation of SK-N-BE(2)c cells with 100 nM CPEC followed by incubation with 500 nM [(3)H]AraC increased the total amount of AraC nucleotides and incorporation of [(3)H]AraC into DNA by 392% and 337%, respectively, compared to non-CPEC-treated cells. When 20 nM [(3)H]AraC was used, the maximum incorporation of [(3)H]AraC into DNA was 1,378% compared to non-CPEC-treated cells. Incorporation of AraC into DNA correlated well with the accumulation of cells in S phase of the cell cycle caused by CPEC. DNA synthesis was almost completely inhibited (>91%) when 100 nM CPEC and 500 nM AraC were combined. CPEC alone and the combination of CPEC and AraC increased caspase-3 activity 3-fold, indicating induction of apoptosis in SK-N-BE(2)c cells. In contrast, AraC alone did not induce caspase-3 activity. Our results demonstrate that low concentrations of CPEC profoundly increase the cytostatic properties of AraC toward SK-N-BE(2)c human neuroblastoma cells.

Expression of the Bacillus subtilis pyrG gene, which encodes CTP synthetase, is repressed by cytidine nucleotides. Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5′ untranslated pyrG leader sequence. Deletion and substitution mutagenesis of a series of pyrG ′ -lacZ transcriptional fusions integrated into the B. subtilis chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of pyrG by starvation for cytidine nucleotides. The first sequence, GGGC/U, comprises the first four nucleotides at the 5′ end of the pyrG transcript, and the second, GCUCCC, forms the first six nucleotides of the 5′ strand of the terminator stem. All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation. We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the pyrG leader region when intracellular CTP levels are low.

The cytotoxic effect of 1-beta-D-arabinofuranosyl cytosine (araC) depends on the intracellular phosphorylation into its active compound araCTP, on the degree of degradation of araCTP and on the incorporation of araCTP into DNA. Deoxycytidine triphosphate (dCTP) inhibits the phosphorylation of araC (by feedback inhibition of the enzyme deoxycytidine kinase) and the incorporation of araCTP into DNA (by competition for DNA polymerase). In a T-lymphoblastic cell line, we studied whether the cytotoxicity of araC (2 nM-50 microM) could be enhanced by decreasing the concentration of dCTP, using the nucleoside-analogue cyclopentenyl cytosine (CPEC), an inhibitor of the enzyme CTP synthetase. Preincubation of the cells with CPEC (100-1,600 nM) for 2 hr increased the concentrations of araCMP 1.6-9.5-fold, which was significant for each concentration of CPEC used. The concentration of araCDP remained low, whereas the concentration of araCTP changed depending on the concentration of araC used. With 2-15 microm of araC and a preincubation with 400 nM of CPEC, the araCTP concentration increased by 4-15% (not significant), and the total amount of araC nucleotides increased significantly by 21-45%. When using a concentration of araC of 2 nM after a preincubation with CPEC of 100 nM, the concentration of araCMP increased by 60% (p = 0.015), whereas that of araCTP decreased by 10% (p = 0.008). This was compensated by an increase of 41% (p = 0.005) of araCTP incorporation into DNA, which represented 43% of all araC metabolites. Moreover, by performing pulse/chase experiments with 400 nM of CPEC and 2 microM of araC, the retention of cytosolic araCTP and the incorporated amount of araCTP into DNA were increased by CPEC. The modulation by CPEC of araC metabolism was accompanied by a synergistic increase of araC-induced apoptosis and by an additive effect on the araC-induced growth inhibition.

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001, eLife digest Enzymes are proteins that perform reactions that can convert one or more chemicals (the substrates) into others (the products). The rate at which an enzyme produces its product is often carefully regulated. Some molecules slow or stop an enzyme by binding to and blocking the site where its substrates normally bind: its ‘active site’. Other molecules can also bind to sites other than the active site, which can cause the enzyme to become either more or less active. Almost all living things have an enzyme called CTP synthetase that makes one of the building blocks that is used to build DNA and a similar molecule called RNA. This enzyme converts a molecule called uridine triphosphate (or UTP) into another called cytidine triphosphate (CTP): a reaction that is powered by breaking down molecules of adenosine triphosphate (ATP). The amount of CTP synthetase made by a cell is carefully controlled. The enzyme's activity is also regulated by the levels of UTP and CTP, and by another molecule (called GTP) that binds to a site outside of its active site. Four copies of the CTP synthetase protein must work together before this enzyme can turn UTP into CTP. The enzyme also forms much larger aggregates, or polymers; however, it is not clear what causes these polymers to form, or what they do in a cell. Barry et al. have now discovered that CTP synthetase is almost completely inactivated when these polymers are formed. Furthermore, CTP encourages the polymers to form, whilst UTP and ATP cause them to disassemble. Therefore, this enzyme is least active when there is excess product in the cell, and most active when its substrates are plentiful. By determining the three-dimensional structure of a CTP synthetase polymer, Barry et al. reveal that although CTP is bound to the enzymes, their active sites are still freely accessible. However, the enzymes in the polymer appear to be locked into a shape that makes them unable to carry out their function. When Barry et al. then mutated the enzyme so that it was unable to form polymers it was also no longer inactivated in the same way by CTP. Bacterial cells with only these mutant versions of CTP synthetase are unable to properly control their levels of CTP. This suggests that polymer formation is important for regulating this enzyme in response to a build up of its product. Further work is needed to see whether the regulation of CTP synthetase activity by forming polymers is specific to this enzyme or a widespread mechanism that is used to control other enzymes too. DOI: http://dx.doi.org/10.7554/eLife.03638.002

The B. subtilis pyrG gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated. The pyrG promoter and 5′ leader were fused to lacZ and integrated into the chromosomes of several B. subtilis strains having mutations in genes of pyrimidine biosynthesis and salvage. These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium. These experiments indicated that pyrG expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides. The start of pyrG transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon. A factor-independent termination hairpin lying between the pyrG promoter and its coding region is essential for regulation of pyrG expression. Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator. Furthermore, deletion of the terminator from pyrG-lacZ fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine. We suggest that cytidine repression of pyrG expression is mediated by an antitermination mechanism in which antitermination by a putative trans -acting protein is reduced by elevated levels of cytidine nucleotides. Conservation of sequences and secondary structural elements in the pyrG 5′ leaders of several other gram-positive bacteria indicates that their pyrG genes are regulated by a similar mechanism.

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei , which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo- l -norleucine (DON) and α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei- infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

A full-length cDNA clone encoding an isoform of human CTP synthetase (type II) was isolated. A 1761-nucleotide open reading frame which corresponds to a protein of 586 amino acids with a predicted molecular mass of 65678 Da was identified. The predicted protein sequence showed 74% identity with the translation product of a previously identified human CTP synthetase cDNA clone (type I). The function of the human cDNA encoding type II CTP synthetase was verified by successful complementation of the cytidine-requiring CTP synthetase deficient mutant JF618 of Escherichia coli. The gene encoding type II CTP synthetase has been localized on chromosome Xp22.

A large-scale production system of cytidine 5'monophospho-N-acetylneuraminic acid (CMP-NeuAc) and sialyloligosaccharides was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammonia-genes. For the production of CMP-NeuAc, two recombinant E. coli strains were generated that overexpressed the genes of CMP-NeuAc synthetase and CTP synthetase, respectively. C. ammoniagenes contributed to the formation of UTP from orotic acid. CMP-NeuAc was accumulated at 27 mM (17 g/l) after a 27-h reaction starting with orotic acid and N-acetylneuraminic acid. When E. coli cells that overexpressed the alpha-(2--3)-sialyltransferase gene of Neisseria gonorrhoeae were put into the CMP-NeuAc production system, 3'-sialyllactose was accumulated at 52 mM (33 g/l) after an 11-h reaction starting with orotic acid, N-acetylneuraminic acid, and lactose. Almost no oligosaccharide byproducts other than 3'-sialyllactose were observed after the reaction. The production of 3'-sialyllactose at a 5-l jar fermenter scale was almost the same as that at a beaker scale, which indicated the high potential of the 3'-sialyllactose production on an industrial scale.