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Protein & Cell May 2023
Topics: Oxidative Phosphorylation; Acetylglucosamine; Uridine Diphosphate N-Acetylglucosamine
PubMed: 37155316
DOI: 10.1093/procel/pwac009 -
Angewandte Chemie (International Ed. in... Aug 2021Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or...
Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
Topics: Animals; Arginine; Cattle; Cross-Linking Reagents; DNA; Histones; Ketones; Peptides; Proteins; Serum Albumin, Bovine; Thymine Nucleotides; Tumor Suppressor Protein p53
PubMed: 34107150
DOI: 10.1002/anie.202105126 -
Annals of Medicine Dec 2023We sought to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in dry eyes treated with 0.05% cyclosporin A and 3.0% diquafosol tetrasodium.
PURPOSE
We sought to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in dry eyes treated with 0.05% cyclosporin A and 3.0% diquafosol tetrasodium.
METHODS
One-hundred ninety-five eyes of 195 patients with dry eye were divided into three groups as follows: group 1, cyclosporin group ( = 69); group 2, diquafosol group ( = 59); and group 3, artificial tears eyes ( = 67). All eyes were treated and followed up for three months. Schirmer I Test, corneal staining, tear-film break-up time (TBUT), and tear-film MMP-9 content were measured at three months and compared between groups. The expression of MMP-9 was confirmed using a point-of-care test device (InflammaDry®; RPS Diagnostics, Sarasota, FL, USA) and graded as zero to four points.
RESULTS
At the third month, MMP-9 expression was lower in group 1 as compared with in groups 2 and 3 ( 0.020 and 0.006, respectively). The mean MMP-9 grade according to point-of-care testing was also lower in group 1 than in groups 2 or 3 ( 0.002 and 0.038, respectively). MMP-9 showed a correlation with corneal staining in both groups 1 and 2 (all < 0.001) and with Schirmer I Test and TBUT in group 1 ( = 0.018 and 0.015, respectively).
CONCLUSIONS
MMP-9 expression and grade were lower after treatment with cyclosporin than after treatment with diquafosol in the dry eye disease. Anti-inflammatory treatment can decrease ocular MMP-9 levels in dry eye disease.
Topics: Humans; Cyclosporine; Matrix Metalloproteinase 9; Dry Eye Syndromes; Uracil Nucleotides
PubMed: 37354028
DOI: 10.1080/07853890.2023.2228192 -
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 -
Chembiochem : a European Journal of... Jan 2022High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the...
High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 μL) within 24 h using 0.02 g /g . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 g /g . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.
Topics: Adenosine Triphosphate; Bioreactors; Cell-Free System; Enzymes; Hydrogen-Ion Concentration; Oligosaccharides; Proof of Concept Study; Uridine Diphosphate Galactose
PubMed: 34637168
DOI: 10.1002/cbic.202100361 -
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 -
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 -
Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase.Scientific Reports Dec 2023Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8...
Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.
Topics: Humans; Galactosyltransferases; Glycosyltransferases; Catalysis; Uridine Diphosphate
PubMed: 38066107
DOI: 10.1038/s41598-023-48605-4