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Cell Reports May 20242'3'-Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) is a second messenger synthesized upon detection of cytosolic double-stranded DNA (dsDNA) and passed between cells...
2'3'-Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) is a second messenger synthesized upon detection of cytosolic double-stranded DNA (dsDNA) and passed between cells to facilitate downstream immune signaling. Ectonucleotide pyrophosphatase phosphodiesterase I (ENPP1), an extracellular enzyme, was the only metazoan hydrolase known to regulate cGAMP levels to dampen anti-cancer immunity. Here, we uncover ENPP3 as the second and likely the only other metazoan cGAMP hydrolase under homeostatic conditions. ENPP3 has a tissue expression pattern distinct from ENPP1's and accounts for all cGAMP hydrolysis activity in ENPP1-deficient mice. Importantly, we also show that, as with ENPP1, selectively abolishing ENPP3's cGAMP hydrolysis activity results in diminished cancer growth and metastasis of certain tumor types in a stimulator of interferon genes (STING)-dependent manner. Both ENPP1 and ENPP3 are extracellular enzymes, suggesting the dominant role that extracellular cGAMP must play as a mediator of cell-cell innate immune communication. Our work demonstrates that ENPP1 and ENPP3 non-redundantly dampen extracellular cGAMP-STING signaling, pointing to ENPP3 as a target for cancer immunotherapy.
Topics: Animals; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Immunity, Innate; Mice; Membrane Proteins; Pyrophosphatases; Humans; Mice, Inbred C57BL; Hydrolysis; Neoplasms; Signal Transduction
PubMed: 38749434
DOI: 10.1016/j.celrep.2024.114209 -
Nucleic Acids Research Jun 2024Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate...
Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.
Topics: Humans; Deoxyguanine Nucleotides; DNA Repair Enzymes; Escherichia coli; Escherichia coli Proteins; Guanine Nucleotides; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Pyrophosphatases
PubMed: 38738661
DOI: 10.1093/nar/gkae371 -
Natural Product Research May 2024is used as a decorative tree and currently studied as a source of biofuels. Besides, its parts and extracts are endowed with several therapeutic uses which have been...
is used as a decorative tree and currently studied as a source of biofuels. Besides, its parts and extracts are endowed with several therapeutic uses which have been widely explored in traditional medicine and that are related to its rich composition in phytochemicals. Molecular docking and enzymatic inhibition tests were used to study the activity of eriodictyol, a flavonoid extracted from the barks of , against ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and aldose reductase (ALR2). The compound was highlighted as a micromolar inhibitor (IC = 263.76 ± 1.32 µM and 4.21 ± 0.94 µM, respectively) and docking showed that eriodictyol efficiently targets the binding sites of the enzymes. In conclusion, this study unveils the potential of eriodictyol on enzymes that are involved in immunostimulation and in complications of diabetes mellitus.
PubMed: 38712509
DOI: 10.1080/14786419.2024.2344744 -
PloS One 2024Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of...
Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.
Topics: Humans; Inorganic Pyrophosphatase; Cell Proliferation; Animals; Carcinoma, Hepatocellular; Liver Neoplasms; Mice; Cell Line, Tumor; Up-Regulation; Xenograft Model Antitumor Assays; Cell Movement; Gene Expression Regulation, Neoplastic; Mice, Nude
PubMed: 38696452
DOI: 10.1371/journal.pone.0299522 -
Biotechnology and Bioengineering May 2024The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling...
The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling of IVT could inform reaction design, scale-up, and control. In this work, we develop a mechanistic model of IVT to include nucleation and growth of magnesium pyrophosphate crystals and subsequent agglomeration of crystals and DNA. To help generalize this model to different constructs, a novel quantitative description is included for the rate of transcription as a function of target sequence length, DNA concentration, and T7 RNA polymerase concentration. The model explains previously unexplained trends in IVT data and quantitatively predicts the effect of adding the pyrophosphatase enzyme to the reaction system. The model is validated on additional literature data showing an ability to predict transcription rates as a function of RNA sequence length.
PubMed: 38695152
DOI: 10.1002/bit.28699 -
Structure (London, England : 1993) Apr 2024Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and...
Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity.
PubMed: 38688286
DOI: 10.1016/j.str.2024.04.006 -
Journal of Cellular and Molecular... May 2024Cisplatin (DDP) resistance is a major challenge in treating ovarian cancer patients. A recently discovered enzyme called dCTP pyrophosphatase 1 (DCTPP1) has been...
Cisplatin (DDP) resistance is a major challenge in treating ovarian cancer patients. A recently discovered enzyme called dCTP pyrophosphatase 1 (DCTPP1) has been implicated in regulating cancer characteristics, including drug responses. In this study, we aimed to understand the role of DCTPP1 in cancer progression and cisplatin response. Using publicly available databases, we analysed the expression and clinical significance of DCTPP1 in ovarian cancer. Our bioinformatics analysis confirmed that DCTPP1 is significantly overexpressed in ovarian cancer and is closely associated with tumour progression and poor prognosis after cisplatin treatment. We also found that DCTPP1 located in oxidoreductase complex and may be involved in various biological processes related to cisplatin resistance, including pyrimidine nucleotide metabolism, the P53 signalling pathway and cell cycle signalling pathways. We observed higher expression of DCTPP1 in cisplatin-resistant cells (SKOV3/DDP) and samples compared to their sensitive counterparts. Additionally, we found that DCTPP1 expression was only enhanced in SKOV3/S cells when treated with cisplatin, indicating different expression patterns of DCTPP1 in cisplatin-sensitive and cisplatin-resistant cancer cells. Our study further supports the notion that cisplatin induces intracellular reactive oxygen species (ROS) and triggers cancer cell death through excessive oxidative stress. Knocking out DCTPP1 reversed the drug resistance of ovarian cancer cells by enhancing the intracellular antioxidant stress response and accumulating ROS. Based on our research findings, we conclude that DCTPP1 has prognostic value for ovarian cancer patients, and targeting DCTPP1 may be clinically significant in overcoming cisplatin resistance in ovarian cancer.
Topics: Humans; Cisplatin; Female; Ovarian Neoplasms; Pyrophosphatases; Drug Resistance, Neoplasm; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Reactive Oxygen Species; Prognosis; Signal Transduction; Antineoplastic Agents
PubMed: 38686496
DOI: 10.1111/jcmm.18371 -
Scientific Reports Apr 2024Aging-related sarcopenia is a degenerative loss of strength and skeletal muscle mass that impairs quality of life. Evaluating NUDT3 gene and myogenin expression as new... (Comparative Study)
Comparative Study
Aging-related sarcopenia is a degenerative loss of strength and skeletal muscle mass that impairs quality of life. Evaluating NUDT3 gene and myogenin expression as new diagnostic tools in sarcopenia. Also, comparing the concomitant treatment of resistance exercise (EX) and creatine monohydrate (CrM) versus single therapy by EX, coenzyme Q10 (CoQ10), and CrM using aged rats. Sixty male rats were equally divided into groups. The control group, aging group, EX-treated group, the CoQ10 group were administered (500 mg/kg) of CoQ10, the CrM group supplied (0.3 mg/kg of CrM), and a group of CrM concomitant with resistance exercise. Serum lipid profiles, certain antioxidant markers, electromyography (EMG), nudix hydrolase 3 (NUDT3) expression, creatine kinase (CK), and sarcopenic index markers were measured after 12 weeks. The gastrocnemius muscle was stained with hematoxylin-eosin (H&E) and myogenin. The EX-CrM combination showed significant improvement in serum lipid profile, antioxidant markers, EMG, NUDT3 gene, myogenin expression, CK, and sarcopenic index markers from other groups. The NUDT3 gene and myogenin expression have proven efficient as diagnostic tools for sarcopenia. Concomitant treatment of CrM and EX is preferable to individual therapy because it reduces inflammation, improves the lipid serum profile, promotes muscle regeneration, and thus has the potential to improve sarcopenia.
Topics: Sarcopenia; Animals; Male; Rats; Creatine; Resistance Training; Muscle, Skeletal; Aging; Physical Conditioning, Animal; Myogenin; Ubiquinone; Pyrophosphatases; Antioxidants; Creatine Kinase; Rats, Wistar
PubMed: 38684784
DOI: 10.1038/s41598-024-59884-w -
Pharmacogenetics and Genomics Jul 2024Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. 6-Mercaptopurine (6-MP) is a key component of ALL treatment. Its use, however, is also...
Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. 6-Mercaptopurine (6-MP) is a key component of ALL treatment. Its use, however, is also associated with adverse drug reactions, particularly myelosuppression. Thiopurine S-methyltransferase (TPMT) and, more recently, Nudix hydrolase 15 (NUDT15) deficiency, due to no-function variants in their respective genes, are well known for their role in the development of this toxicity. Two novel genetic variants, rs12199316 in TPMT and rs73189762 in the NUDT15 gene, were recently identified by targeted sequencing. The latter is particularly interesting because of its potential association with 6-MP intolerance. Here, we assessed the relationship of this variant with the risk of myelosuppression and 6-MP dose intensity in 275 patients treated with Dana Farber Cancer Institute ALL protocols at the Sainte Justine University Health Center. Carriers of the NUDT15 rs73189762 variant allele were at a higher risk of myelosuppression, as shown by absolute phagocyte count reduction during consolidation II and maintenance phases of therapy. Reduction in 6-MP dose intensity was observed in patients with both rs73189762 and known no-function variants in the NUDT15 and TPMT genes. This finding supports the initial observation and suggests that 6-MP dose reduction might be beneficial for individuals with this genotype combination.
Topics: Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrophosphatases; Child; Male; Female; Child, Preschool; Adolescent; Antimetabolites, Antineoplastic; Methyltransferases; Infant; Polymorphism, Single Nucleotide; Nudix Hydrolases
PubMed: 38682355
DOI: 10.1097/FPC.0000000000000533 -
Analytica Chimica Acta May 2024Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways...
BACKGROUND
Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis.
RESULT
The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu) ions. The off state of LA@AuAg_Cu ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu ions, and again quenched off state was retrieved due to interaction of free Cu with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples.
SIGNIFICANCE
The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.
Topics: Copper; Gold; Inorganic Pyrophosphatase; Silver; Metal Nanoparticles; Fluorometry; Diphosphates; Humans; Limit of Detection; Infrared Rays
PubMed: 38677840
DOI: 10.1016/j.aca.2024.342584