Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Adrenal pheochromocytoma impacts three main pathways: cysteine-methionine, pyrimidine, and tyrosine metabolism. / è‚¾ä¸Šè ºå—œé“¬ç»†èƒžç˜¤å½±å“ä¸‰æ¡ä¸»è¦ä»£è°¢é€šè·¯ï¼šåŠèƒ±æ°¨é ¸-è›‹æ°¨é ¸ä»£è°¢ã€å˜§å•¶ä»£è°¢å’Œé ªæ°¨é ¸ä»£è°¢é€šè·¯.

Pheochromocytomas and paragangliomas (PPGLs) cause symptoms by altering the circulation levels of catecholamines and peptide hormones. Currently, the diagnosis of PPGLs relies on diagnostic imaging and the detection of catecholamines. In this study, we used ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight mass spectrometry (Q-TOF MS) analysis to identify and measure the perioperative differential metabolites in the plasma of adrenal pheochromocytoma patients. We identified differentially expressed genes by comparing the transcriptomic data of pheochromocytoma with the normal adrenal medulla. Through conducting two steps of metabolomics analysis, we identified 111 differential metabolites between the healthy group and the patient group, among which 53 metabolites were validated. By integrating the information of differential metabolites and differentially expressed genes, we inferred that the cysteine-methionine, pyrimidine, and tyrosine metabolism pathways were the three main metabolic pathways altered by the neoplasm. The analysis of transcription levels revealed that the tyrosine and cysteine-methionine metabolism pathways were downregulated in pheochromocytoma, whereas the pyrimidine pathway showed no significant difference. Finally, we developed an optimized diagnostic model of two metabolites, L-dihydroorotic acid and vanylglycol. Our results for these metabolites suggest that they may serve as potential clinical biomarkers and can be used to supplement and improve the diagnosis of pheochromocytoma.

Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Biochemical and structural characterization of Fapy•dG replication by Human DNA polymerase ß.

N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway.

Background: Amino acid metabolism, including ATP production, nucleotide synthesis, and redox homeostatic processes, are associated with proliferation and differentiation of tumor cells. This study aimed to identify novel prognostic biomarkers and potential therapeutic targets of amino acid metabolism-related genes for stomach adenocarcinoma (STAD). Methods: RNA sequencing transcriptome data in the TCGA-STAD (training set) and GTEx datasets (validation set) were used. The LIMMA R program enabled the differentially expressed amino acid metabolism-related genes (AAMRGs) to be found. A prognostic risk score model based on clinical phenotypic features was built using LASSO regression and step multi-Cox analyses. Gene set enrichment analysis (GSEA) was used to find potential molecular pathways associated with STAD. Hierarchical cluster analysis was used to evaluate pyrimidine metabolism. Cultured STAD cells assessed the proliferation of STAD and upregulation of GPX3 expression by CCK8 and flow cytometry. Transwell and wound healing assays assessed the impact of GPX3 on invasion and migration of STAD cells. Western blot and qRT-PCR were used to measure changes in pyrimidine metabolism-related markers and active molecules involved in the AMPK/mTOR signaling pathway. Results: Three AAMRGs, DNMT1, F2R, and GPX3, could independently predict the course of STAD. Pyrimidine metabolism appeared to be significantly associated with these by GSEA and clustering analyses. Pyrimidine metabolism was negatively correlated with GPX3. Functional studies using an overexpressed GPX3 plasmid showed an enhanced migration and invasion of STAD cells as well as the expression of genes associated with pyrimidine metabolism and the AMPK/mTOR signaling pathway. By using a CAD siRNA, it was found that that GPX3 affected 5-fluorouracil resistance during de novo synthesis of pyrimidine through the CAD-UMPS signaling axis. Conclusions: GPX3 which regulates the level of pyrimidine metabolism through the AMPK/mTOR pathway was found to be closely associated with STAD. Our findings demonstrate GPX3 is a reliable biomarker for the prognosis of amino acid metabolism and a probable target for STAD therapy.

Nitrogen assimilation by E. coli in the mammalian intestine.

Nitrogen is an essential element for all living organisms, including Escherichia coli. Potential nitrogen sources are abundant in the intestine, but knowledge of those used specifically by E. coli to colonize remains limited. Here, we sought to determine the specific nitrogen sources used by E. coli to colonize the streptomycin-treated mouse intestine. We began by investigating whether nitrogen is limiting in the intestine. The NtrBC two-component system upregulates approximately 100 genes in response to nitrogen limitation. We showed that NtrBC is crucial for E. coli colonization, although most genes of the NtrBC regulon are not induced, which indicates that nitrogen is not limiting in the intestine. RNA-seq identified upregulated genes in colonized E. coli involved in transport and catabolism of seven amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine. Competitive colonization experiments revealed that L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides serve as nitrogen sources for E. coli in the intestine. Furthermore, the colonization defect of a L-serine deaminase mutant was rescued by excess nitrogen in the drinking water but not by an excess of carbon and energy, demonstrating that L-serine serves primarily as a nitrogen source. Similar rescue experiments showed that N-acetylneuraminic acid serves as both a carbon and nitrogen source. To a minor extent, aspartate and ammonia also serve as nitrogen sources. Overall, these findings demonstrate that E. coli utilizes multiple nitrogen sources for successful colonization of the mouse intestine, the most important of which is L-serine. IMPORTANCE: While much is known about the carbon and energy sources that are used by E. coli to colonize the mammalian intestine, very little is known about the sources of nitrogen. Interrogation of colonized E. coli by RNA-seq revealed that nitrogen is not limiting, indicating an abundance of nitrogen sources in the intestine. Pathways for assimilation of nitrogen from several amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine were induced in mice. Competitive colonization assays confirmed that mutants lacking catabolic pathways for L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides had colonization defects. Rescue experiments in mice showed that L-serine serves primarily as a nitrogen source, whereas N-acetylneuraminic acid provides both carbon and nitrogen. Of the many nitrogen assimilation mutants tested, the largest colonization defect was for an L-serine deaminase mutant, which demonstrates L-serine is the most important nitrogen source for colonized E. coli.

ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Neurological presentations of inborn errors of purine and pyrimidine metabolism.

Purines and pyrimidines are essential components as they are the building blocks of vital molecules, such as nucleic acids, coenzymes, signalling molecules, as well as energy transfer molecules. Purine and pyrimidine metabolism defects are characterised by abnormal concentrations of purines, pyrimidines and/or their metabolites in cells or body fluids. This phenomenon is due to a decreased or an increased activity of enzymes involved in this metabolism and has been reported in humans for over 60 years. This review provides an overview of neurological presentations of inborn errors of purine and pyrimidine metabolism. These conditions can lead to psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscular symptoms. Clinical signs are often nonspecific and thus overlooked, but some diseases are treatable and early diagnosis may improve the child's future. Although these metabolic hereditary diseases are rare, they are most probably under-diagnosed. When confronted with suggestive clinical or laboratory signs, clinicians should prescribe genetic testing in association with a biochemical screening including thorough purine and pyrimidine metabolites analysis and/or specific enzyme evaluation. This is most likely going to increase the number of confirmed patients.

mRNA incorporation of C(5)-halogenated pyrimidine ribonucleotides and induced high expression of corresponding protein for the development of mRNA vaccine.

In this report, we present our studies on mRNA, which was modified by introducing various halogen substituents at the C(5) position of the pyrimidine base. Specifically, we synthesized C(5)-halogenated (F, Cl, Br, I) pyrimidine ribonucleoside triphosphates and incorporated them into mRNA during in-vitro transcription. The efficiency of the in-vitro transcription reaction of halogenated pyrimidine was observed to decrease as the size of the halogen substituent increased and the electronegativity thereof decreased (F > Cl > Br) except for iodine. Interestingly, we found that, among the C(5)-halogenated pyrimidine ribonucleotides, mRNA incorporating C(5)-halogenated cytidine (5-F rCTP and 5-Cl rCTP) exhibited more prominent protein expression than mRNA modified with C(5)-halogenated uridine and unmodified mRNA. In particular, in the case of mRNA to which fluorine (5-F rCTP) and chlorine (5-Cl rCTP) were introduced, the protein was dramatically expressed about 4 to 5 times more efficiently than the unmodified mRNA, which was similar to pseudouridine (ψ). More interestingly, when pseudouridine(ψ) and fluorocytidine nucleotides (5-F rCTP), were simultaneously introduced into mRNA for dual incorporation, the protein expression efficiency dramatically increased as much as tenfold. The efficiency of cap-dependent protein expression is much higher than the IRES-dependent (internal ribosome entry site) expression with mRNA incorporating C(5)-halogenated pyrimidine ribonucleotide. We expect these results to contribute meaningfully to the development of therapeutics based on modified mRNA.

Blockade of de novo pyrimidine biosynthesis triggers autophagic degradation of oncoprotein FLT3-ITD in acute myeloid leukemia.

The internal tandem duplication of the FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Limited and transient clinical benefit of FLT3 kinase inhibitors (FLT3i) emphasizes the need for alternative therapeutic options for this subset of myeloid malignancies. Herein, we showed that FLT3-ITD mutant (FLT3-ITD+) AML cells were susceptible toward inhibitors of DHODH, a rate-limiting enzyme of de novo pyrimidine biosynthesis. Genetic and pharmacological blockade of DHODH triggered downregulation of FLT3-ITD protein, subsequently suppressed activation of downstream ERK and STAT5, and promoted cell death of FLT3-ITD+ AML cells. Mechanistically, DHODH blockade triggered autophagy-mediated FLT3-ITD degradation via inactivating mTOR, a potent autophagy repressor. Notably, blockade of DHODH synergized with an FDA-approved FLT3i quizartinib in significantly impairing the growth of FLT3-ITD+ AML cells and improving tumor-bearing mice survival. We further demonstrated that DHODH blockade exhibited profound anti-proliferation effect on quizartinib-resistant cells in vitro and in vivo. In summary, this study demonstrates that the induction of degradation of FLT3-ITD protein by DHODH blockade may offer a promising therapeutic strategy for AML patients harboring FLT3-ITD mutation.

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract.

Among eukaryotes, the major spliceosomal pathway is highly conserved. While long introns may contain additional regulatory sequences, the ones in short introns seem to be nearly exclusively related to splicing. Although these regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3' end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines, and the polypyrimidine tract upstream of this 3' splice signal is characterized by over-representation of pyrimidines. If the over-representation of pyrimidines in the polypyrimidine tract is also due to avoidance of a premature splicing signal, we hypothesize that AG should be the most under-represented dimer. Through the use of DNA-strand asymmetry patterns, we confirm this prediction in fruit flies of the genus Drosophila and by comparing the asymmetry patterns to a presumably neutrally evolving region, we quantify the selection strength acting on each motif. Moreover, our inference and simulation method revealed that the best explanation for the base composition evolution of the polypyrimidine tract is the joint action of purifying selection against a spurious 3' splice signal and the selection for pyrimidines. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.

Discovery of potent and selective HPK1 inhibitors based on the 2,4-disubstituted pyrimidine scaffold with immune modulatory properties for ameliorating T cell exhaustion.

Hematopoietic progenitor kinase 1 (HPK1), a member of the mitogen-activated protein kinase (MAP4K) family, is a serine/threonine (SER/THR) kinase and has been demonstrated as a negative regulator of T cell receptor signaling. Targeting HPK1 has been considered as an attractive therapeutic strategy for immune-oncology. Here, we describe the discovery and structure-activity relationship (SAR) of potent HPK1 inhibitors based on the 2,4-disubstituted pyrimidine scaffold. Systematically SAR exploration afforded the desired compound HMC-H8 (F1) with potent HPK1 inhibition (IC50 = 1.11 nM) and highly selectivity profile. Compound HMC-H8 also exhibited robust inhibition of p-SLP 76 (IC50 = 283.0 nM) and promotion IL-2 release (EC50 = 157.08 nM), and INF-γ production in a dose-dependent manner in vitro assays. Strikingly, HMC-H8 shown effective immune reversal response in immunesuppressive condition. Moreover, Compound HMC-H8 displayed acceptable metabolic stability (T1/2 = 56.87 min), along with low CYP450 inhibition in human liver microsomes and good oral bioavailability (F = 15.05%) in rat. Furthermore, HMC-H8 was found to modulate the expression of c-Myc in Western blotting experiments. Taken together, this study provides new potent HPK1 inhibitors for further anticancer drug discovery based on immuno-oncology.

Structure-Activity Relationship Studies of 2,4,5-Trisubstituted Pyrimidine Derivatives Leading to the Identification of a Novel and Potent Sirtuin 5 Inhibitor against Sepsis-Associated Acute Kidney Injury.

Sepsis-associated acute kidney injury (AKI) is a serious clinical problem without effective drugs. Inhibition of sirtuin 5 (SIRT5) has been confirmed to protect against AKI, suggesting that SIRT5 inhibitors might be a promising therapeutic approach for AKI. Herein, structural optimization was performed on our previous compound 1 (IC50 = 3.0 µM), and a series of 2,4,5-trisubstituted pyrimidine derivatives have been synthesized. The structure-activity relationship (SAR) analysis led to the discovery of three nanomolar level SIRT5 inhibitors, of which the most potent compound 58 (IC50 = 310 nM) was demonstrated to be a substrate-competitive and selective inhibitor. Importantly, 58 significantly alleviated kidney dysfunction and pathological injury in both lipopolysaccharide (LPS)- and cecal ligation/perforation (CLP)-induced septic AKI mice. Further studies revealed that 58 regulated protein succinylation and the release of proinflammatory cytokines in the kidneys of septic AKI mice. Collectively, these results highlighted that targeting SIRT5 has a therapeutic potential against septic AKI.

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression.

Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.

Pyrimidine catabolism is required to prevent the accumulation of 5-methyluridine in RNA.

5-Methylated cytosine is a frequent modification in eukaryotic RNA and DNA influencing mRNA stability and gene expression. Here we show that free 5-methylcytidine (5mC) and 5-methyl-2'-deoxycytidine are generated from nucleic acid turnover in Arabidopsis thaliana, and elucidate how these cytidines are degraded, which is unclear in eukaryotes. First CYTIDINE DEAMINASE produces 5-methyluridine (5mU) and thymidine which are subsequently hydrolyzed by NUCLEOSIDE HYDROLASE 1 (NSH1) to thymine and ribose or deoxyribose. Interestingly, far more thymine is generated from RNA than from DNA turnover, and most 5mU is directly released from RNA without a 5mC intermediate, since 5-methylated uridine (m5U) is an abundant RNA modification (m5U/U ∼1%) in Arabidopsis. We show that m5U is introduced mainly by tRNA-SPECIFIC METHYLTRANSFERASE 2A and 2B. Genetic disruption of 5mU degradation in the NSH1 mutant causes m5U to occur in mRNA and results in reduced seedling growth, which is aggravated by external 5mU supplementation, also leading to more m5U in all RNA species. Given the similarities between pyrimidine catabolism in plants, mammals and other eukaryotes, we hypothesize that the removal of 5mU is an important function of pyrimidine degradation in many organisms, which in plants serves to protect RNA from stochastic m5U modification.

Impact of Negative Feedbacks on De Novo Pyrimidines Biosynthesis in Escherichia coli.

Earlier studies aimed at investigating the metabolism of endogenous nucleoside triphosphates in synchronous cultures of E. coli cells revealed an auto-oscillatory mode of functioning of the pyrimidine and purine nucleotide biosynthesis system, which the authors associated with the dynamics of cell division. Theoretically, this system has an intrinsic oscillatory potential, since the dynamics of its functioning are controlled through feedback mechanisms. The question of whether the nucleotide biosynthesis system has its own oscillatory circuit is still open. To address this issue, an integral mathematical model of pyrimidine biosynthesis was developed, taking into account all experimentally verified negative feedback in the regulation of enzymatic reactions, the data of which were obtained under in vitro conditions. Analysis of the dynamic modes of the model functioning has shown that in the pyrimidine biosynthesis system, both the steady-state and oscillatory functioning modes can be realized under certain sets of kinetic parameters that fit in the physiological boundaries of the investigated metabolic system. It has been demonstrated that the occurrence of the oscillatory nature of metabolite synthesis depended on the ratio of two parameters: the Hill coefficient, hUMP1-the nonlinearity of the UMP effect on the activity of carbamoyl-phosphate synthetase, and the parameter r characterizing the contribution of the noncompetitive mechanism of UTP inhibition to the regulation of the enzymatic reaction of UMP phosphorylation. Thus, it has been theoretically shown that the E. coli pyrimidine biosynthesis system possesses its own oscillatory circuit whose oscillatory potential depends to a significant degree on the mechanism of regulation of UMP kinase activity.

Nucleotide Imbalance, Provoked by Downregulation of Aspartate Transcarbamoylase Impairs Cold Acclimation in Arabidopsis.

Aspartate transcarbamoylase (ATC) catalyzes the first committed step in pyrimidine de novo synthesis. As shown before, mutants with 80% reduced transcript and protein levels exhibit reduced levels of pyrimidine metabolites and thus nucleotide limitation and imbalance. Consequently, reduced photosynthetic capacity and growth, accompanied by massive transcriptional changes, were observed. Here, we show that nucleotide de novo synthesis was upregulated during cold acclimation of Arabidopsis thaliana (ecotype Columbia, Col-0) plants, but ATC knockdown mutants failed to acclimate to this condition as they did not accumulate neutral sugars and anthocyanins. A global transcriptome analysis revealed that most of the transcriptional changes observed in Col-0 plants upon cold exposure were also evident in ATC knockdown plants. However, several responses observed in cold-treated Col-0 plants could already be detected in knockdown plants when grown under standard conditions, suggesting that these mutants exhibited typical cold responses without prior cold stimulation. We believe that nucleotide signaling is involved in "cold-like priming" and "cold acclimation" in general. The observed transcript levels of genes involved in central carbon metabolism and respiration were an exception to these findings. These were upregulated in the cold but downregulated in warm-grown ATC mutants.