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Trends in Biochemical Sciences Jan 2015The cyclic purine nucleotides cAMP and cGMP are established second messengers. By contrast, the existence of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic...
The cyclic purine nucleotides cAMP and cGMP are established second messengers. By contrast, the existence of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) has been controversial for decades. The recent development of highly sensitive mass spectrometry (MS) methods allowed precise quantitation and unequivocal identification of cCMP and cUMP in cells. Importantly, cCMP and cUMP generators, effectors, cleaving enzymes, and transporters have now been identified. Here, I discuss evidence in support of cCMP and cUMP as bona fide second messengers, the emerging therapeutic implications of cCMP and cUMP signaling, and important unresolved questions for this field.
Topics: Adenylyl Cyclases; Bacterial Proteins; Bacterial Toxins; Cyclic CMP; Glucosyltransferases; Guanylate Cyclase; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Protein Kinases; Pseudomonas aeruginosa; Uridine Monophosphate
PubMed: 25435399
DOI: 10.1016/j.tibs.2014.10.008 -
ACS Chemical Biology Oct 2022Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine,...
Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
Topics: Pyrimidine Nucleotides; 5-Methylcytosine; DNA-Directed DNA Polymerase; Nucleotides; DNA; DNA Restriction Enzymes; Pyrimidines; Deoxyribonucleosides; Epigenesis, Genetic
PubMed: 35679536
DOI: 10.1021/acschembio.2c00342 -
Stem Cell Research & Therapy Apr 2023Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation...
BACKGROUND
Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs.
METHODS
MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3 antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y purinoceptors prevented this effect.
CONCLUSIONS
Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.
Topics: Humans; Female; Aged; Adolescent; Young Adult; Adult; Osteogenesis; Postmenopause; Mesenchymal Stem Cells; Cell Differentiation; Uracil Nucleotides; Uridine Diphosphate; Adenosine Triphosphate; Bone Marrow Cells; Cells, Cultured
PubMed: 37076930
DOI: 10.1186/s13287-023-03315-6 -
Nature Communications May 2021High-yielding and selective prebiotic syntheses of RNA and DNA nucleotides involve UV irradiation to promote the key reaction steps and eradicate biologically irrelevant...
High-yielding and selective prebiotic syntheses of RNA and DNA nucleotides involve UV irradiation to promote the key reaction steps and eradicate biologically irrelevant isomers. While these syntheses were likely enabled by UV-rich prebiotic environment, UV-induced formation of photodamages in polymeric nucleic acids, such as cyclobutane pyrimidine dimers (CPDs), remains the key unresolved issue for the origins of RNA and DNA on Earth. Here, we demonstrate that substitution of adenine with 2,6-diaminopurine enables repair of CPDs with yields reaching 92%. This substantial self-repairing activity originates from excellent electron donating properties of 2,6-diaminopurine in nucleic acid strands. We also show that the deoxyribonucleosides of 2,6-diaminopurine and adenine can be formed under the same prebiotic conditions. Considering that 2,6-diaminopurine was previously shown to increase the rate of nonenzymatic RNA replication, this nucleobase could have played critical roles in the formation of functional and photostable RNA/DNA oligomers in UV-rich prebiotic environments.
Topics: 2-Aminopurine; Adenine; DNA; DNA Repair; Molecular Dynamics Simulation; Nucleic Acids; Nucleotides; Pyrimidine Dimers; RNA; Ultraviolet Rays
PubMed: 34021158
DOI: 10.1038/s41467-021-23300-y -
Archives of Biochemistry and Biophysics Oct 2019In view of previous crystallographic studies, N-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate...
In view of previous crystallographic studies, N-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
Topics: Animals; Deoxycytidine Monophosphate; Enzyme Inhibitors; Fluorodeoxyuridylate; Folic Acid; Mice; Protein Binding; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Thymidylate Synthase
PubMed: 31520592
DOI: 10.1016/j.abb.2019.108106 -
The FEBS Journal Oct 2018Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis...
Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP-tagged IMPDH2 and GFP-tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super-resolution confocal imaging, we demonstrate how IMPDH- and CTPS-based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.
Topics: Carbon-Nitrogen Ligases; Cytidine Triphosphate; Cytoskeleton; Genes, Reporter; HeLa Cells; Humans; IMP Dehydrogenase; Microscopy, Confocal
PubMed: 30085408
DOI: 10.1111/febs.14624 -
Angewandte Chemie (International Ed. in... Oct 2021Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami...
Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.
Topics: DNA; DNA Nucleotidylexotransferase; Deoxyuracil Nucleotides; Nanostructures; Nucleic Acid Conformation; Polymerization; Polynucleotides; Proof of Concept Study; Thymine Nucleotides
PubMed: 34302317
DOI: 10.1002/anie.202107829 -
Carbohydrate Research Sep 2020UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported...
UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro substitutions. A concise chemoenzymatic synthesis was developed using the kinase NahK (B. longum JCM1217) and the uridyl transferase GlmU (E. coli K12).
Topics: Carbohydrate Conformation; Uridine Diphosphate N-Acetylglucosamine
PubMed: 32634644
DOI: 10.1016/j.carres.2020.108071 -
Microcirculation (New York, N.Y. : 1994) Jan 2018Smooth muscle voltage-gated potassium (Kv) channels are important regulators of microvascular tone and tissue perfusion. Recent studies indicate that Kv1 channels... (Review)
Review
Smooth muscle voltage-gated potassium (Kv) channels are important regulators of microvascular tone and tissue perfusion. Recent studies indicate that Kv1 channels represent a key component of the physiological coupling between coronary blood flow and myocardial oxygen demand. While the mechanisms by which metabolic changes in the heart are transduced to alter coronary Kv1 channel gating and promote vasodilation are unclear, a growing body of evidence underscores a pivotal role of Kv1 channels in sensing the cellular redox status. Here, we discuss current knowledge of mechanisms of Kv channel redox regulation with respect to pyridine nucleotide modulation of Kv1 function via ancillary Kvβ proteins as well as direct modulation of channel activity via reactive oxygen and nitrogen species. We identify areas of additional research to address the integration of regulatory processes under altered physiological and pathophysiological conditions that may reveal insights into novel treatment strategies for conditions in which the matching of coronary blood supply and myocardial oxygen demand is compromised.
Topics: Animals; Coronary Vessels; Humans; Microcirculation; Oxidation-Reduction; Potassium Channels, Voltage-Gated; Pyrimidine Nucleotides; Reactive Nitrogen Species; Reactive Oxygen Species
PubMed: 29110409
DOI: 10.1111/micc.12426 -
Cancer Chemotherapy and Pharmacology Jun 2020In preclinical models of biliary tract cancer, NUC-1031 showed less potency than gemcitabine, no correlation with potential biomarkers and only moderate additive...
In preclinical models of biliary tract cancer, NUC-1031 showed less potency than gemcitabine, no correlation with potential biomarkers and only moderate additive interaction in combination with cisplatin. These findings should prompt further careful pharmacological and translational studies to better define the purported therapeutic advantage of NUC-1031 over gemcitabine. That would be a more cautious approach than the phase III clinical trial which is planning to enrol 828 patients with biliary tract tumours to compare gemcitabine/cisplatin "conventional" treatment with or without NUC-1031.
Topics: Antineoplastic Agents; Biliary Tract Neoplasms; Cytidine Monophosphate; Humans; Prognosis; Translational Research, Biomedical
PubMed: 32476108
DOI: 10.1007/s00280-020-04080-6