Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities.
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ABSTRACT:
SUBMITTER: Baykov AA
PROVIDER: S-EPMC8073611 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity.ImportanceRepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.
| S-EPMC11323792 | biostudies-literature
Project description:FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC(50). The assays were optimized to a low 4?µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC(50) of 0.59 and 0.93?µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target.
| S-EPMC3025571 | biostudies-literature
Project description:IntroductionThe physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.MethodsWe treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.ResultsInorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.ConclusionsIncreased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).
| S-EPMC2818162 | biostudies-literature
Project description:cDNA corresponding to two type-I vacuolar H(+)-inorganic pyrophosphatases (V-PPases) (SlVP1, SlVP2) and one type-II V-PPase (SlVP3) was isolated from tomato fruit to investigate their role in fruit development. Southern analysis revealed that type-I V-PPase genes form a multigene family, whereas there is only one type-II V-PPase gene in the tomato genome. Although SlVP1 and SlVP2 were differentially expressed in leaves and mature fruit, the highest levels of both SlVP1 and SlVP2 mRNA were observed in fruit at 2-4 days after anthesis. The expression pattern of type-II SlVP3 was similar to that of SlVP2, and the highest levels of SlVP3 mRNA were also observed in fruit at 2-4 days after anthesis, thus suggesting that SlVP3 plays a role in early fruit development. Because SlVP1 and SlVP2 mRNA was more abundant than SlVP3 mRNA, expression of type-I V-PPases was analysed further. Type-I V-PPase mRNA was localized in ovules and their vicinities and in vascular tissue at an early stage of fruit development. Tomato RNAi lines in which the expression of type-I V-PPase genes was repressed using the fruit-specific promoter TPRP-F1 exhibited fruit growth retardation at an early stage of development. Although the major function of V-PPases in fruit has been believed to be the accumulation of materials such as sugars and organic acids in the vacuole during cell expansion and ripening, these results show that specific localization of V-PPase mRNA induced by pollination has a novel role in the cell division stage.
| S-EPMC3444275 | biostudies-literature
Project description:The present work is focused on testing enzyme-based agents for the partial dissolution of calcium pyrophosphate (CaPPi) deposits in the cartilages and synovial fluid of patients with pyrophosphate arthropathy (CPPD disease). Previously, we suggested that inorganic pyrophosphatases (PPases) immobilized on nanodiamonds of detonation synthesis (NDs) could be appropriate for this purpose. We synthesized and characterized conjugates of NDs and PPases from Escherichia coli and Mycobacterium tuberculosis. The conjugates showed high enzymatic activity and resistance to inhibition by calcium and fluoride. Here, we tested the effectiveness of pyrophosphate (PPi) hydrolysis by the conjugates in an in vitro model system simulating the ionic composition of the synovial fluid and in the samples of synovial fluid of patients with CPPD via NMR spectroscopy. The conjugates of both PPases efficiently hydrolyzed triclinic crystalline calcium pyrophosphate (t-CPPD) in the model system. We evaluated the number of phosphorus-containing compounds in the synovial fluid, showed the possibility of PPi detection in it, and estimated the hydrolytic activity of the PPase conjugates. The soluble and immobilized PPases were able to hydrolyze a significant amount of PPi (1 mM) in the synovial fluid in short periods of time (24 h). The maximum activity was demonstrated for Mt-PPase immobilized on ND-NH-(CH2)6-NH2 (2.24 U mg-1).
| S-EPMC7178341 | biostudies-literature
Project description:Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase-pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z' = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.
| S-EPMC3575809 | biostudies-literature
Project description:1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.
| S-EPMC1187773 | biostudies-other
Project description:Tumor cells rely on aerobic glycolysis to support growth and survival, thus require more glucose supply. Glucose transporters GLUTs, primarily GLUT1, are overexpressed in various cancers. Targeting GLUTs has been regarded as a promising anticancer strategy. In this study, we first evaluated 75 potential GLUT1 inhibitors obtained from virtual screening of the NCI chemical library by a high-throughput cell-based method using a fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) in COS-7 and SKOV3 cells that express high levels of GLUT1. Four compounds, #12, #16, #43 and #69, that significantly inhibited glucose uptake were further evaluated using flow cytometry directly measuring 2-NBDG uptake at the single-cell level and a Glucose Uptake-GloTM assay indirectly measuring 2-deoxy-d-glucose uptake in SKOV3, COS-7 or MCF-7 cells. The inhibitory effect on cancer cell growth was also determined in SKOV3 and MCF-7 cells, and #12 exhibited the best growth inhibitory effect equivalent to a known GLUT1 inhibitor WZB117. Although the anticancer effect of the identified potential GLUT1 inhibitors was moderate, they may enhance the activity of other anticancer drugs. Indeed, we found that #12 synergistically enhanced the anticancer activity of metformin in SKOV3 ovarian cancer cells.
| S-EPMC9692963 | biostudies-literature
Project description:Pyrophosphate arthropathy is the mineralization defect in humans caused by the deposition of microcrystals of calcium pyrophosphate dihydrate in joint tissues. As a potential therapeutic strategy for the treatment of pyrophosphate arthropathy, delivery of exogenous pyrophosphate-hydrolyzing enzymes, inorganic pyrophosphatases (PPases), to the synovial fluid has been suggested. Previously, we synthesized the conjugates of Escherichia coli PPase (Ec-PPase) with detonation synthesis nanodiamonds (NDs) as a delivery platform, obtaining the hybrid biomaterial retaining high pyrophosphate-hydrolyzing activity in vitro. However, most known PPases including Ec-PPase in the soluble form are strongly inhibited by Ca2+ ions. Because synovial fluid contains up to millimolar concentrations of soluble calcium, this inhibition might limit the in vivo application of Ec-PPase-based material in joint tissues. In this work, we proposed other bacterial PPases from Mycobacterium tuberculosis (Mt-PPase), which are resistant to the inhibition by Ca2+ ions, as an active PPi-hydrolyzing agent. We synthesized conjugates of Mt-PPase with NDs and tested their activity under various conditions. Unexpectedly, conjugates of both Ec-PPase and Mt-PPase with aminated NDs retained significant hydrolytic activity in the presence of well-known mechanism-based PPase inhibitors, fluoride or calcium. The incomplete inhibition of PPases by fluoride or calcium was found for the first time.
| S-EPMC7114608 | biostudies-literature
Project description: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.
| S-EPMC10311840 | biostudies-literature