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Nature Jun 2013Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host...
Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2'-5'oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.
Topics: Adenosine Triphosphate; Animals; Base Sequence; Catalytic Domain; Crystallography, X-Ray; Cytosol; DNA; Guanosine Triphosphate; HEK293 Cells; Humans; Membrane Proteins; Mice; Models, Biological; Models, Molecular; Mutation; Nucleotidyltransferases; Protein Conformation; Structure-Activity Relationship; Substrate Specificity; Swine; Uridine Triphosphate; Zinc
PubMed: 23722159
DOI: 10.1038/nature12305 -
FASEB Journal : Official Publication of... Jan 2016Adenosine (ADO) and nucleotides such as ATP, ADP, and uridine 5'-triphosphate (UTP), among others, may serve as extracellular signaling molecules. These mediators... (Review)
Review
Adenosine (ADO) and nucleotides such as ATP, ADP, and uridine 5'-triphosphate (UTP), among others, may serve as extracellular signaling molecules. These mediators activate specific cell-surface receptors-namely, purinergic 1 and 2 (P1 and P2)-to modulate crucial pathophysiological responses. Regulation of this process is maintained by nucleoside and nucleotide transporters, as well as the ectonucleotidases ectonucleoside triphosphate diphosphohydrolase [ENTPD; cluster of differentiation (CD)39] and ecto-5'-nucleotidase (5'-NT; CD73), among others. Cells involved in tissue repair, healing, and scarring respond to both ADO and ATP. Our recent investigations have shown that modulation of purinergic signaling regulates matrix deposition during tissue repair and fibrosis in several organs. Cells release adenine nucleotides into the extracellular space, where these mediators are converted by CD39 and CD73 into ADO, which is anti-inflammatory in the short term but may also promote dermal, heart, liver, and lung fibrosis with repetitive signaling under defined circumstances. Extracellular ATP stimulates cardiac fibroblast proliferation, lung inflammation, and fibrosis. P2Y2 (UTP/ATP) and P2Y6 [ADP/UTP/uridine 5'-diphosphate (UDP)] have been shown to have profibrotic effects, as well. Modulation of purinergic signaling represents a novel approach to preventing or diminishing fibrosis. We provide an overview of the current understanding of purinergic signaling in scarring and discuss its potential to prevent or decrease fibrosis.
Topics: Adenosine; Animals; Fibrosis; Humans; Liver; Purinergic Agents; Signal Transduction; Uridine Triphosphate
PubMed: 26333425
DOI: 10.1096/fj.15-274563 -
Life Science Alliance Sep 2023The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and...
The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of and/or and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.
Topics: Carbon-Nitrogen Ligases; Uridine Triphosphate; Cell Proliferation; Cell Cycle; Cell Line
PubMed: 37348953
DOI: 10.26508/lsa.202302066 -
International Journal of Molecular... Jan 2017Platelet P2Y is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development...
Platelet P2Y is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y but not P2Y₁ receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC value of ~250 °µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2-UTP), with an IC of 30 µM. The 4-thio analogue (4-UTP) with an IC of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y receptor was verified by P2Y receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y receptor antagonists that may be useful for therapeutic intervention.
Topics: Adenosine Triphosphate; Adult; Blood Platelets; Cell Adhesion Molecules; Cyclic AMP; Female; Healthy Volunteers; Humans; Male; Microfilament Proteins; Middle Aged; Molecular Structure; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet-Rich Plasma; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Uridine Triphosphate; Young Adult
PubMed: 28146050
DOI: 10.3390/ijms18020269 -
Nucleic Acids Research Oct 2022In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA...
In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5'-AAAU-3' RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3' extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an 'open' to a 'closed' state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.
Topics: Arabidopsis; Arabidopsis Proteins; MicroRNAs; Purines; RNA Nucleotidyltransferases; RNA, Messenger; Uridine Triphosphate
PubMed: 36177876
DOI: 10.1093/nar/gkac839 -
American Journal of Physiology. Renal... Sep 2011Extracellular nucleotides (e.g., ATP) activate ionotropic P2X and metabotropic P2Y receptors in the plasma membrane to regulate and maintain cell function and integrity.... (Review)
Review
Extracellular nucleotides (e.g., ATP) activate ionotropic P2X and metabotropic P2Y receptors in the plasma membrane to regulate and maintain cell function and integrity. This includes the renal tubular and collecting duct system, where the locally released nucleotides act in a paracrine and autocrine way to regulate transport of electrolytes and water and maintain cell volume. A prominent role has been assigned to Gq-coupled P2Y(2) receptors, which are typically activated by both ATP and UTP. Studies in gene knockout mice revealed an antihypertensive activity of P2Y(2) receptors that is linked to vasodilation and an inhibitory influence on renal salt reabsorption. Flow induces apical ATP release in the thick ascending limb, and first evidence indicates an inhibitory influence of P2Y(2) receptor tone on the expression and activity of the Na-K-2Cl cotransporter NKCC2 in this segment. The apical ATP/UTP/P2Y(2) receptor system in the connecting tubule/cortical collecting duct mediates the inhibitory effect of dietary salt on the open probability of the epithelial sodium channel ENaC and inhibits ENaC activity during aldosterone escape. Connexin 30 has been implicated in the luminal release of the ATP involved in the regulation of ENaC. An increase in collecting duct cell volume in response to manipulating water homeostasis increases ATP release. The subsequent activation of P2Y(2) receptors inhibits vasopressin-induced cAMP formation and water reabsorption, which facilitates water excretion and stabilizes cell volume. Thus recent studies have established the ATP/UTP/P2Y(2) receptor system as a relevant regulator of renal salt and water homeostasis and blood pressure regulation. The pathophysiological relevance and therapeutic potential remains to be determined, but dual effects of P2Y(2) receptor activation on both the vasculature and renal salt reabsorption implicate these receptors as potential therapeutic targets in hypertension.
Topics: Adenosine Triphosphate; Animals; Biological Transport; Kidney; Mice; Mice, Knockout; Models, Animal; Receptors, Purinergic P2Y2; Signal Transduction; Sodium Chloride; Uridine Triphosphate; Water
PubMed: 21715471
DOI: 10.1152/ajprenal.00236.2011 -
Biochemistry Feb 2020Viperin is a radical -adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by...
Viperin is a radical -adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, is similar for CTP and UTP whereas the for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.
Topics: Animals; Crystallography, X-Ray; Cytidine Triphosphate; Kinetics; Mice; Models, Molecular; Molecular Structure; Mutation; Proteins; S-Adenosylhomocysteine; Substrate Specificity; Uridine Triphosphate
PubMed: 31917549
DOI: 10.1021/acs.biochem.9b00741 -
Nucleic Acids Research Sep 2020Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction....
Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.
Topics: Fluorescein; Fluorescent Dyes; Genome, Mitochondrial; High-Throughput Screening Assays; Multiplex Polymerase Chain Reaction; Protozoan Proteins; RNA Editing; RNA, Guide, Kinetoplastida; RNA, Messenger; Trypanosoma brucei brucei; Uridine Triphosphate
PubMed: 32756897
DOI: 10.1093/nar/gkaa658 -
Biomolecules Apr 2022Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP.... (Review)
Review
Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
Topics: Adenosine Triphosphate; Allosteric Regulation; Carbon-Nitrogen Ligases; Cryoelectron Microscopy; Cytidine Triphosphate; Glutaminase; Glutamine; Guanosine Triphosphate; Nitric Oxide Synthase; Uridine Triphosphate
PubMed: 35625575
DOI: 10.3390/biom12050647 -
Complementary Therapies in Medicine Aug 2022Bailing Capsule (BLC), Jinshuibao (JSB), Huangkui Capsule (HKC), Uremic Clearance Granule (UCG), Tripterygium glycosides (TG), Compound Xueshuantong Capsule (CXC), and... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
Bailing Capsule (BLC), Jinshuibao (JSB), Huangkui Capsule (HKC), Uremic Clearance Granule (UCG), Tripterygium glycosides (TG), Compound Xueshuantong Capsule (CXC), and Shenyan Kangfu Tablet (SYKFT) as classic Chinese patent medicines (CPMs), have been widely used and shown beneficial effects on the treatment of early diabetic kidney disease (DKD). However, the comparative efficacy of seven CPMs in the treatment of early DKD remains unknown.
OBJECTIVE
To evaluate and compare the efficacy of seven CPMs (BLC, JSB, HKC, UCG, TG, CXC, SYKFT) combined with angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) on early DKD by a Bayesian network meta-analysis (NMA) of randomized controlled trials (RCTs).
METHODS
A comprehensive and systematic literature search was performed in PubMed, Embase, Cochrane Library, Web of Science, Clinical Trials.gov, China Biology Medicine, Chinese National Knowledge Infrastructure, Chinese Scientific Journal, and Wanfang databases from inception to March 14, 2021, for full-text RCTs that evaluated the efficacy of seven CPMs combined with ACEI/ARB on patients with early DKD. Two reviewers independently screened studies for eligibility, extracted data, and assessed the risk of bias. Agreement between reviewers was measured using kappa statistics. Mean difference (MD) and odds ratio (OR) were calculated to evaluate continuous variables and dichotomous, respectively. The random effect modeling NMA was performed and the ranking probability of interventions in various outcomes was also conducted based on the surface under the cumulative ranking curve (SUCRA). Begg's and Egger's tests were used to evaluate publication bias. The certainty of the evidence for outcomes was evaluated according to the GRADE system.
RESULTS
A total of 62 RCTs with 5362 patients with early DKD were identified. The value of Kappa calculated for the various parameters extracted by the two investigators was 0.821 (P < 0.001). Among these CPMs, UCG + ACEI/ARB showed the best effectiveness for urinary albumin excretion rate (UAER) (MD 32.25, 95% CrI 19.11-45.67, low certainty) with SUCRA 92%. JSB + ACEI/ARB showed the highest effectiveness for 24-h urinary total protein (24-h UTP) (MD 76.92, 95% CrI 53.54-100.58, low certainty) with SUCRA 97%. CXC + ACEI/ARB showed the highest effectiveness for serum creatinine (SCr) (MD 26.02, 95% CrI 6.10-45.95, low certainty) with SUCRA 96%. HKC + ACEI/ARB showed the highest effectiveness for blood urea nitrogen (BUN) (MD 1.46, 95% CrI 0.42-2.54, very low certainty) with SUCRA 86%. BLC + ACEI/ARB showed significant differences in triglyceride (TRIG) (MD - 1.17, 95% CrI - 1.93 to - 0.43, low certainty) with SUCRA 90%, total cholesterol (TC) (MD - 1.17, 95% CrI - 1.97 to - 0.39, very low certainty) with SUCRA 90%, and C-reaction protein (CRP) (MD - 0.90, 95% CrI - 1.51 to - 0.32, very low certainty) with SUCRA 76%.
CONCLUSIONS
CPMs + ACEI/ARB might be positive efficacious interventions from which patients with DKD will derive benefit. UCG + ACEI/ARB, JSB + ACEI/ARB, CXC + ACEI/ARB, and HKC + ACEI/ARB might be potentially the preferred intervention for reducing UAER, 24-h UTP, SCr, and BUN levels, respectively. BLC + ACEI/ARB has a better impact on lowing TRIG, TC, and CRP levels in patients with early DKD. However, more high-quality, large-scale, multi-center RCTs and stronger head-to-head trials are required to confirm these findings.
Topics: Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Diabetes Mellitus; Diabetic Nephropathies; Female; Humans; Male; Network Meta-Analysis; Nonprescription Drugs; Uridine Triphosphate
PubMed: 35398481
DOI: 10.1016/j.ctim.2022.102831