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Journal of Bone and Mineral Research :... Sep 2022Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of...
Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1 mice. In contrast to Enpp1 mice, which lack ENPP1, Enpp1 mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1 mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear β-catenin signaling compared to wild-type (WT) and Enpp1 cultures. Finally, the decreased calcification and nuclear β-catenin signaling observed in Enpp1 cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).
Topics: Animals; Bone and Bones; Catalysis; Familial Hypophosphatemic Rickets; Fibroblast Growth Factors; Mammals; Mice; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification; beta Catenin
PubMed: 35773783
DOI: 10.1002/jbmr.4640 -
MBio Feb 2022Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription...
Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription interference. The growth defects (ranging from sickness to lethality) elicited by fission yeast mutations that inactivate IPP pyrophosphatase enzymes are exerted via the agonistic effects of too much 1,5-IP8 on RNA 3'-processing and transcription termination. To illuminate determinants of IPP toxicosis, we conducted a genetic screen for spontaneous mutations that suppressed the sickness of Asp1 pyrophosphatase mutants. We identified a missense mutation, C823R, in the essential Cft1 subunit of the cleavage and polyadenylation factor complex that suppresses even lethal Asp1 IPP pyrophosphatase mutations, thereby fortifying the case for 3'-processing/termination as the target of IPP toxicity. The suppressor screen also identified Gde1 and Spx1 (SPAC6B12.07c), both of which have an IPP-binding SPX domain and both of which are required for lethality elicited by Asp1 mutations. A survey of other SPX proteins in the proteome identified the Vtc4 and Vtc2 subunits of the vacuolar polyphosphate polymerase as additional agents of IPP toxicosis. Gde1, Spx1, and Vtc4 contain enzymatic modules (glycerophosphodiesterase, RING finger ubiquitin ligase, and polyphosphate polymerase, respectively) fused to their IPP-sensing SPX domains. Structure-guided mutagenesis of the IPP-binding sites and the catalytic domains of Gde1 and Spx1 indicated that both modules are necessary to elicit IPP toxicity. Whereas Vtc4 polymerase catalytic activity is required for IPP toxicity, its IPP-binding site is not. Epistasis analysis, transcriptome profiling, and assays of Pho1 expression implicate Spx1 as a transducer of IP8 signaling to the 3'-processing/transcription termination machinery. Impeding the catabolism of the inositol pyrophosphate (IPP) signaling molecule IP8 is cytotoxic to fission yeast. Here, by performing a genetic suppressor screen, we identified several cellular proteins required for IPP toxicosis. Alleviation of IPP lethality by a missense mutation in the essential Cft1 subunit of the cleavage and polyadenylation factor consolidates previous evidence that toxicity results from IP8 action as an agonist of RNA 3'-processing and transcription termination. Novel findings are that IP8 toxicity depends on IPP-sensing SPX domain proteins with associated enzymatic functions: Gde1 (glycerophosphodiesterase), Spx1 (ubiquitin ligase), and Vtc2/4 (polyphosphate polymerase). The effects of Spx1 deletion on phosphate homeostasis imply a role for Spx1 in communicating an IP8-driven signal to the transcription and RNA processing apparatus.
Topics: Diphosphates; Fungal Proteins; Inositol Phosphates; Ligases; mRNA Cleavage and Polyadenylation Factors; Polyphosphates; Pyrophosphatases; RNA; Schizosaccharomyces; Ubiquitins
PubMed: 35012333
DOI: 10.1128/mbio.03476-21 -
Scientific Reports Jul 2017Inorganic pyrophosphatases (PPase) participate in energy cycling and they are essential for growth and survival of organisms. Here we report extensive structural and...
Inorganic pyrophosphatases (PPase) participate in energy cycling and they are essential for growth and survival of organisms. Here we report extensive structural and functional characterization of soluble PPases from the human parasites Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase). Our results show that PfPPase is a cytosolic enzyme whose gene expression is upregulated during parasite asexual stages. Cambialistic PfPPase actively hydrolyzes linear short chain polyphosphates like PP, polyP and ATP in the presence of Zn. A remarkable new feature of PfPPase is the low complexity asparagine-rich N-terminal region that mediates its dimerization. Deletion of N-region has an unexpected and substantial effect on the stability of PfPPase domain, resulting in aggregation and significant loss of enzyme activity. Significantly, the crystal structures of PfPPase and TgPPase reveal unusual and unprecedented dimeric organizations and provide new fundamental insights into the variety of oligomeric assemblies possible in eukaryotic inorganic PPases.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Cytosol; Inorganic Pyrophosphatase; Models, Molecular; Phosphotransferases; Plasmodium falciparum; Protein Conformation; Protein Domains; Protein Multimerization; Sequence Homology; Toxoplasma
PubMed: 28701714
DOI: 10.1038/s41598-017-05234-y -
ENPP1, an Old Enzyme with New Functions, and Small Molecule Inhibitors-A STING in the Tale of ENPP1.Molecules (Basel, Switzerland) Nov 2019Ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) was identified several decades ago as a type II transmembrane glycoprotein with nucleotide pyrophosphatase and... (Review)
Review
Ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) was identified several decades ago as a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase enzymatic activities, critical for purinergic signaling. Recently, ENPP1 has emerged as a critical phosphodiesterase that degrades the stimulator of interferon genes (STING) ligand, cyclic GMP-AMP (cGAMP). cGAMP or analogs thereof have emerged as potent immunostimulatory agents, which have potential applications in immunotherapy. This emerging role of ENPP1 has placed this "old" enzyme at the frontier of immunotherapy. This review highlights the roles played by ENPP1, the mechanism of cGAMP hydrolysis by ENPP1, and small molecule inhibitors of ENPP1 with potential applications in diverse disease states, including cancer.
Topics: Animals; Drug Discovery; Gene Expression Regulation; Humans; Hydrolysis; Membrane Proteins; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Protein Binding; Pyrophosphatases; Signal Transduction; Structure-Activity Relationship
PubMed: 31752288
DOI: 10.3390/molecules24224192 -
PLoS Computational Biology Oct 2022Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the...
Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.
Topics: Cations; Cell Membrane; Diphosphates; Lipids; Pyrophosphatases
PubMed: 36191052
DOI: 10.1371/journal.pcbi.1010578 -
SLAS Discovery : Advancing Life... Jun 2021The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor,...
The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP/GMP Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP/GMP Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP/GMP Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.
Topics: Drug Discovery; Fluorescence Polarization Immunoassay; High-Throughput Screening Assays; Humans; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 33402044
DOI: 10.1177/2472555220982321 -
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 -
Metabolic Syndrome and Related Disorders Mar 2017Gestational diabetes mellitus (GDM) is a metabolic disorder characterized by insulin resistance (IR) and altered glucose-lipid metabolism. We propose that ectonucleotide...
BACKGROUND
Gestational diabetes mellitus (GDM) is a metabolic disorder characterized by insulin resistance (IR) and altered glucose-lipid metabolism. We propose that ectonucleotide pyrophosphate phosphodiesterase-1 (ENPP1), a protein known to induce adipocyte IR, is a determinant of GDM. Our objective was to study ENPP1 expression in adipose tissue (AT) of obese pregnant women with or without GDM, as well as glucose tolerance in pregnant transgenic (Tg) mice with AT-specific overexpression of human ENPP1.
METHODS
AT biopsies and blood were collected from body mass index-matched obese pregnant women non-GDM (n = 6), GDM (n = 7), and nonpregnant controls (n = 6) undergoing cesarian section or elective surgeries, respectively. We measured the following: (1) Expression of key molecules involved in insulin signaling and glucose-lipid metabolism in AT; (2) Plasma glucose and insulin levels and calculation of homeostasis model assessment of IR (HOMA-IR); (3) Intraperitoneal glucose tolerance test in AtENPP1 Tg pregnant mice.
RESULTS
We found that: (1) Obese GDM patients have higher AT ENPP1 expression than obese non-GDM patients, or controls (P = 0.01-ANOVA). (2) ENPP1 expression level correlated negatively with glucose transporter 4 (GLUT4) and positively with insulin receptor substrate-1 (IRS-1) serine phosphorylation, and to other adipocyte functional proteins involved in glucose and lipid metabolism (P < 0.05 each), (3) AT ENPP1 expression levels were positively correlated with HOMA-IR (P = 0.01-ANOVA). (4) Pregnant AT ENPP1 Tg mice showed higher plasma glucose than wild type animals (P = 0.046-t test on area under curve [AUC]).
CONCLUSIONS
Our results provide evidence of a causative link between ENPP1 and alterations in insulin signaling, glucose uptake, and lipid metabolism in subcutaneous abdominal AT of GDM, which may mediate IR and hyperglycemia in GDM.
Topics: Adipose Tissue; Adult; Animals; Case-Control Studies; Cross-Sectional Studies; Diabetes, Gestational; Female; Humans; Insulin Resistance; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphoric Diester Hydrolases; Pregnancy; Pyrophosphatases; Signal Transduction
PubMed: 28080219
DOI: 10.1089/met.2016.0124 -
The Journal of Molecular Diagnostics :... Oct 2022Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are...
Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.
Topics: Alleles; DNA; Genetic Testing; Haplotypes; Humans; Methyltransferases; Pharmacogenetics; Pyrophosphatases
PubMed: 35931342
DOI: 10.1016/j.jmoldx.2022.06.008 -
Kidney International Jul 2018Protein carbamylation is a posttranslational modification that can occur non-enzymatically in the presence of high concentrations of urea. Although carbamylation is...
Protein carbamylation is a posttranslational modification that can occur non-enzymatically in the presence of high concentrations of urea. Although carbamylation is recognized as a prognostic biomarker, the contribution of protein carbamylation to organ dysfunction remains uncertain. Because vascular calcification is common under carbamylation-prone situations, we investigated the effects of carbamylation on this pathologic condition. Protein carbamylation exacerbated the calcification of human vascular smooth muscle cells (hVSMCs) by suppressing the expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1), a key enzyme in the generation of pyrophosphate, which is a potent inhibitor of ectopic calcification. Several mitochondrial proteins were carbamylated, although ENPP1 itself was not identified as a carbamylated protein. Rather, protein carbamylation reduced mitochondrial membrane potential and exaggerated mitochondria-derived oxidative stress, which down-regulated ENPP1. The effects of carbamylation on ectopic calcification were abolished in hVSMCs by ENPP1 knockdown, in mitochondrial-DNA-depleted hVSMCs, and in hVSMCs treated with a mitochondria-targeted superoxide scavenger. We also evaluated the carbamylation effects using ex vivo and in vivo models. The tunica media of a patient with end-stage renal disease was carbamylated. Thus, our findings have uncovered a previously unrecognized aspect of uremia-related vascular pathology.
Topics: Animals; Cell Line; Disease Models, Animal; Disease Progression; Gene Knockdown Techniques; Humans; Kidney Failure, Chronic; Male; Membrane Potential, Mitochondrial; Muscle, Smooth, Vascular; Oxidative Stress; Phosphoric Diester Hydrolases; Protein Carbamylation; Pyrophosphatases; Rats; Rats, Sprague-Dawley; Uremia; Vascular Calcification
PubMed: 29716796
DOI: 10.1016/j.kint.2018.01.033