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European Journal of Biochemistry Apr 1989Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human...
Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins.
Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.
Topics: Cell Aggregation; Enzyme Activation; Female; GTP-Binding Proteins; Glucuronidase; Humans; In Vitro Techniques; Kinetics; Male; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Purine Nucleotides; Pyrimidine Nucleotides; Ribonucleotides; Structure-Activity Relationship; Superoxides
PubMed: 2540969
DOI: 10.1111/j.1432-1033.1989.tb14722.x -
European Journal of Biochemistry Feb 1985Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed...
Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed information on the specificity of nucleotide-enzyme interaction. The 1H investigations focussed on those protons presumed to be involved in hydrogen bonding between the various nucleotides and the enzyme. In particular these were the imino protons of the uridine nucleotides and the amino protons of the cytidine nucleotides. The technique of 15N-1H double quantum filtering was applied for observation of the resonances of the latter in the nucleotide-enzyme complex. The downfield shift observed for the imino proton resonance of the uridine nucleotides was indicative of hydrogen bond formation to the enzyme. 15N NMR spectra of the free nucleotides and the nucleotide-enzyme complexes were also acquired to examine the possibility of hydrogen bond formation at the N3 site of both pyrimidine bases and the amino group of the cytidine nucleotides. The downfield shift observed for the 15N3 resonance of the uridine nucleotides and the upfield shift observed for the corresponding resonance of the cytidine nucleotides was evidence that the N3 moiety acts as hydrogen donor or hydrogen acceptor in the nucleotide-enzyme complex. The effect of complex formation on the 15N1 resonance of the respective bases was also studied. Both 1H and 15N NMR results indicated subtle differences between the complexes of the 2' and 3' nucleotides. The extent of hydrogen bonding as well as the arrangement of the nucleotide base at the active site of the enzyme varies in dependence on the position of the phosphate group. It is established that hydrogen bonding, though not the main binding force between the nucleotides and the enzyme, is certainly a major factor of RNase A specificity for pyrimidine nucleotides.
Topics: Chemical Phenomena; Chemistry; Hydrogen; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Protein Binding; Pyrimidine Nucleotides; Ribonuclease, Pancreatic
PubMed: 3971970
DOI: 10.1111/j.1432-1033.1985.tb08708.x -
Chembiochem : a European Journal of... Jul 2014Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR...
Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).
Topics: Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Pyrimidine Nucleotides; RNA; Stereoisomerism
PubMed: 24954297
DOI: 10.1002/cbic.201402130 -
Proceedings of the National Academy of... Nov 2019If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and...
If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at ATTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers.
Topics: 5' Untranslated Regions; Cells, Cultured; DNA Damage; Fibroblasts; Gene Expression Regulation; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Melanocytes; Melanoma; Mutation; Promoter Regions, Genetic; Protein Biosynthesis; Pyrimidine Dimers; Pyrimidine Nucleotides; Skin Neoplasms; TOR Serine-Threonine Kinases; Ultraviolet Rays
PubMed: 31723047
DOI: 10.1073/pnas.1907860116 -
Molecular Pharmacology Mar 2009Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF),... (Comparative Study)
Comparative Study
Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides.
Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides ( Mol Pharmacol 70: 878-886, 2006 ). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2'(3')-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (K(i), 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 is crucial for FRET to MANT-ATP and MANT-CTP and that the mutations N583Q, K353A, and K353R differentially alter the inhibitory potencies of MANT-ATP and MANT-CTP. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors.
Topics: Adenosine Diphosphate; Adenylyl Cyclases; Adenylyl Imidodiphosphate; Animals; Anthrax Vaccines; Antigens, Bacterial; Bacterial Toxins; Catalysis; Cattle; Crystallography, X-Ray; Fluorescence Resonance Energy Transfer; Mutagenesis, Site-Directed; Protein Binding; Purine Nucleotides; Pyrimidine Nucleotides; ortho-Aminobenzoates
PubMed: 19056899
DOI: 10.1124/mol.108.052340 -
Blood Advances Mar 2024Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine...
Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.
Topics: Adult; Humans; Pyrimidines; Uridine; Cell Proliferation; Cytidine; Human T-lymphotropic virus 1; Pyrimidine Nucleotides; Receptors, Antigen, T-Cell; T-Lymphocytes
PubMed: 38190613
DOI: 10.1182/bloodadvances.2023011131 -
The Biochemical Journal Jan 2006Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In...
Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization.
Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent K(m) of 207 microM for TTP, 404 microM for UTP and 435 microM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.
Topics: Biological Transport; Carrier Proteins; Cloning, Molecular; Escherichia coli; Gene Expression; Kinetics; Mitochondria; Mitochondrial Proteins; Nucleotide Transport Proteins; Pyrimidine Nucleotides; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 16194150
DOI: 10.1042/BJ20051284 -
Journal of the American Chemical Society Aug 2022Although 2'-deoxy-2'-α-F-2'-β--methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in...
Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-β--methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.
Although 2'-deoxy-2'-α-F-2'-β--methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'- preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary -acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both and isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'--methyl, the isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the -methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.
Topics: Animals; Liposomes; Mice; Models, Molecular; Nanoparticles; Nucleic Acid Conformation; Nucleosides; Nucleotides; Oligonucleotides; Organophosphonates; Phosphates; Pyrimidine Nucleotides; RNA Interference; RNA, Small Interfering
PubMed: 35921401
DOI: 10.1021/jacs.2c01679 -
British Journal of Pharmacology Dec 19931. Using a grease-gap technique, we have investigated the effects of purine and pyrimidine nucleotides on the d.c. potential of the rat isolated superior cervical...
1. Using a grease-gap technique, we have investigated the effects of purine and pyrimidine nucleotides on the d.c. potential of the rat isolated superior cervical ganglion (SCG). 2. Of the purines tested, adenosine, adenosine 5'-triphosphate (ATP), beta,gamma-methylene-adenosine 5'-triphosphate (beta,gamma-MeATP) at up to 300 microM produced concentration-dependent hyperpolarizations, whereas 2-methyl-thio-ATP (2-Me.S.ATP) and alpha,beta-methylene-ATP (alpha,beta-MeATP) depolarized ganglia. Of the pyrimidines tested, uridine 5'-triphosphate (UTP) produced concentration-dependent depolarizations and cytosine 5'-triphosphate (CTP) at 1000 microM produced considerably smaller but significant depolarizations. In contrast uridine 5'-monophosphate (UMP) at 1000 microM hyperpolarized ganglia. The relative order of potency of purines and pyrimidines to depolarize ganglia was: UTP > alpha,beta-MeATP >> CTP > 2-Me.S.ATP and to hyperpolarize ganglia was: adenosine = beta,gamma-MeATP > ATP > UMP. 3. The ability of purines and pyrimidines to alter the depolarizing response caused by muscarine and of purines to alter depolarization induced by gamma-aminobutyric acid (GABA) was determined. The relative order of potency of nucleotides in depressing submaximal depolarization caused by muscarine (100 nM) was: adenosine = ATP > beta,gamma-MeATP whereas 2-Me.S.ATP, alpha,beta-MeATP and UTP did not significantly alter depolarization caused by muscarine. At 100 microM beta,gamma-MeATP and adenosine but not ATP potentiated GABA-induced depolarizations. 4. Hyperpolarizations caused by adenosine, ATP, beta,gamma-MeATP and UMP and depolarizations caused by alpha,beta-MeATP were enhanced in medium containing reduced concentrations of calcium (0.1 mM) and potassium (2 mM). In this medium 8-phenyltheophylline abolished hyperpolarizations caused by adenosine and reversed hyperpolarizations caused by ATP into depolarizations. Suramin (300 microM), a P2-purinoceptor antagonist, significantly reduced the depolarizing response caused by alpha,beta-MeATP and significantly increased hyperpolarizations caused by ATP and Beta,gamma-MeATP. Suramin (300 microM) did not significantly alter depolarizations caused by l,l-dimethyl-4-phenylpiperazinium (10 microM), potassium(3 mM) or muscarine (100 nM) and significantly potentiated depolarizations caused by UTP (100 microM).5.It is concluded that the rat SCG contains PI-purinoceptors that hyperpolarize the ganglion and diminish sensitivity to muscarine, and P2X-purinoceptors that depolarize the SCG. There is also some evidence to suggest the presence of receptors for UTP, i.e., pyrimidinoceptors, which depolarize SCG neurones.
Topics: Adenosine Triphosphate; Animals; Calcium; In Vitro Techniques; Male; Membrane Potentials; Purine Nucleotides; Pyrimidine Nucleotides; Rats; Rats, Wistar; Receptors, Purinergic; Superior Cervical Ganglion; Uridine Triphosphate; gamma-Aminobutyric Acid
PubMed: 8306068
DOI: 10.1111/j.1476-5381.1993.tb13959.x -
Purinergic Signalling Dec 2021Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major...
Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the K value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.
Topics: 5'-Nucleotidase; Adenosine; Humans; Hydrolysis; Protein Binding; Protein Folding; Purines; Pyrimidine Nucleotides; Signal Transduction
PubMed: 34403084
DOI: 10.1007/s11302-021-09802-w