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Structure (London, England : 1993) Dec 1996Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these... (Review)
Review
Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these proteins complexed with substrate analogs reveal the structural basis for uracil recognition and suggest a novel mechanism of DNA repair.
Topics: Binding Sites; DNA; DNA Glycosylases; DNA Repair; Deoxyuridine; Models, Molecular; N-Glycosyl Hydrolases; Nucleic Acid Conformation; Protein Conformation; Pyrophosphatases; Uracil; Uracil-DNA Glycosidase
PubMed: 8994964
DOI: 10.1016/s0969-2126(96)00145-1 -
Acta Crystallographica. Section F,... Nov 2018Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length...
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI cells, purified using two chromatographic steps and crystallized. Its structure at 2.77 Å resolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not interact with the PDE domain but extends further away from the PDE domain. Deletion of the SMB domains resulted in crystals that diffracted to 2.4 Å resolution and are suitable for substrate-binding studies.
Topics: Binding Sites; Catalytic Domain; Crystallization; Crystallography, X-Ray; HEK293 Cells; Humans; Phosphoric Diester Hydrolases; Protein Domains; Pyrophosphatases; Recombinant Proteins; Somatomedins
PubMed: 30387774
DOI: 10.1107/S2053230X18011111 -
Microbiology and Molecular Biology... Jun 2013In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate... (Review)
Review
In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H(+) transport across biological membranes (H(+)-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na(+) (Na(+)-pyrophosphatase) or both Na(+) and H(+) (Na(+),H(+)-pyrophosphatase). Both these transporters require Na(+) for pyrophosphate hydrolysis and are further activated by K(+). The determination of the three-dimensional structures of H(+)- and Na(+)-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.
Topics: Animals; Biological Transport; Cation Transport Proteins; Cell Membrane; Diphosphates; Hydrolysis; Phylogeny; Prokaryotic Cells; Protons; Pyrophosphatases; Sodium
PubMed: 23699258
DOI: 10.1128/MMBR.00003-13 -
Biochemistry and Cell Biology =... Oct 2022Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription...
Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.
Topics: Diphosphates; Inorganic Pyrophosphatase; Magnesium; Pyrophosphatases; RNA
PubMed: 35926232
DOI: 10.1139/bcb-2022-0118 -
Cancer Discovery May 2022Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified...
ABSTRACT
Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.
SIGNIFICANCE
CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.
Topics: Breast Neoplasms; Female; Haptoglobins; Humans; Myeloid-Derived Suppressor Cells; Neoplasm Recurrence, Local; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 35191482
DOI: 10.1158/2159-8290.CD-21-0932 -
Biochimica Et Biophysica Acta May 2003The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a... (Review)
Review
The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.
Topics: Amino Acid Sequence; Animals; Calcification, Physiologic; Catalytic Domain; Diabetes Mellitus, Type 2; Gene Expression Regulation, Enzymologic; Humans; Models, Biological; Multigene Family; Neoplasm Invasiveness; Nucleotides; Phosphoric Diester Hydrolases; Phylogeny; Pyrophosphatases; Signal Transduction; Substrate Specificity; Tissue Distribution
PubMed: 12757929
DOI: 10.1016/s0925-4439(03)00058-9 -
Journal of Lipid Research Jan 2016The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX... (Review)
Review
The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX is expressed in multiple tissues and participates in numerous key physiologic and pathologic processes, including neural development, obesity, inflammation, and oncogenesis, through the generation of the bioactive lipid, lysophosphatidic acid. Overwhelming evidence indicates that altered ATX activity leads to oncogenesis and cancer progression through the modulation of multiple hallmarks of cancer pathobiology. Here, we review the structural and catalytic characteristics of the ectoenzyme, how its expression and maturation processes are regulated, and how the systemic integration of its pleomorphic effects on cells and tissues may contribute to cancer initiation, progression, and therapy. Additionally, the up-to-date spectrum of the most frequent ATX genomic alterations from The Cancer Genome Atlas project is reported for a subset of cancers.
Topics: Animals; Carcinogenesis; Cell Transformation, Neoplastic; Disease Progression; Humans; Neoplasms; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 25977291
DOI: 10.1194/jlr.R060020 -
Mutation Research 2013Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to... (Review)
Review
Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
Topics: Animals; Escherichia coli; Humans; Mice; Models, Molecular; Pharmacogenetics; Polymorphism, Genetic; Pyrophosphatases; Yeasts
PubMed: 23969025
DOI: 10.1016/j.mrrev.2013.08.001 -
Nature Chemical Biology Oct 2020The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites,...
The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
Topics: Binding Sites; Cell Line; Drug Design; Drug Development; Escherichia coli; Humans; Inorganic Pyrophosphatase; Models, Molecular; Protein Binding; Protein Conformation; Pyrophosphatases; Structure-Activity Relationship
PubMed: 32690945
DOI: 10.1038/s41589-020-0592-z -
FEBS Letters Sep 1999The earliest known H+-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H+-PPi synthase (proton-pumping inorganic pyrophosphate synthase)... (Review)
Review
The earliest known H+-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H+-PPi synthase (proton-pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-PPi synthase and two algal vacuolar H+-PPases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-PPases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-PPases are reviewed and compared with H+-ATPases and soluble PPases.
Topics: Amino Acid Sequence; Cell Membrane; Evolution, Molecular; Inorganic Pyrophosphatase; Molecular Sequence Data; Plants; Prokaryotic Cells; Pyrophosphatases; Rhodospirillum rubrum; Sequence Homology, Amino Acid; Vacuoles
PubMed: 10523139
DOI: 10.1016/s0014-5793(99)90617-8