Publications

• Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

  Nguyen Trung M., Kieninger S., Fandi Z., Qiu D., Liu G., Mehendale N.K., Saiardi A., Jessen H., Keller B., Fiedler D.

  The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize ... >> More

  The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically ¹³C-labeled <i>myo</i>-inositol and inositol phosphates. These probes were utilized in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to investigate InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR-down to individual enantiomers-which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme contributing to cellular homeostasis. Detailed characterization of MINPP1 activity in vitro and in cells showcased the unique reactivity of this phosphatase. Our results demonstrate that metabolic labeling with stable isotopomers in conjunction with NMR spectroscopy and CE-MS constitutes a powerful tool to annotate InsP networks in a variety of biological contexts. << Less

  ACS Cent. Sci. 8:1683-1694(2022) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.

• Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase.

  Craxton A., Caffrey J.J., Burkhart W., Safrany S.T., Shears S.B.

  The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted a ... >> More

  The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the beta-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167]. << Less

  Biochem. J. 328:75-81(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Purification of an inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity from rat liver and the evaluation of its substrate specificity.

  Nogimori K., Hughes P.J., Glennon M.C., Hodgson M.E., Putney J.W. Jr., Shears S.B.

  Hepatic inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity was detected in a 100,000 x g soluble fraction and a detergent-solubilized particulate fraction. Activity in both fractions increased up to 40-fold after anion-exchange chromatography due to removal of endogenous inhibitors (Hodgs ... >> More

  Hepatic inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity was detected in a 100,000 x g soluble fraction and a detergent-solubilized particulate fraction. Activity in both fractions increased up to 40-fold after anion-exchange chromatography due to removal of endogenous inhibitors (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834); at this stage the detergent-solubilized particulate activity comprised over 90% of total activity. The particulate phosphatase was further purified by affinity chromatography using heparin-agarose and red-agarose. The latter column resolved two peaks of enzyme activity (designated 1 and 2 by their order of elution from the column). Their proportions varied between experiments, but peak 2 generally predominated and so this was further purified by hydroxylapatite chromatography. The final preparation was typically 38,000-fold purified with a 7% yield. The apparent molecular mass of this enzyme was 66 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The enzyme had little or no affinity for the following: inositol (1,3,4,6)-tetrakisphosphate, inositol (1,3,4)-trisphosphate, inositol (1,3)-bisphosphate, inositol (3,4)-bisphosphate, and para-nitrophenylphosphate. At pH 7.4 the Km for inositol (1,3,4,5)-tetrakisphosphate was 130 nM and the Vmax was 4250 nmol/mg protein/min. The purified enzyme also dephosphorylated inositol (1,3,4,5,6)-pentakisphosphate to inositol (1,4,5,6)-tetrakisphosphate (Km = 40 nM, Vmax = 211 nmol/mg protein/min), and inositol hexakisphosphate to at least five isomers of inositol pentakisphosphate (Km = 0.3 nM, Vmax = 12 nmol/mg protein/min). The latter affinity is the highest yet defined for an enzyme involved in inositol phosphate metabolism. Determinations of IC50 values, and Dixon plots, revealed that with the (1,3,4,5)-tetrakisphosphate as substrate, the pentakis- and hexakisphosphates were potent competitive inhibitors; the Ki values (25 and 0.5 nM, respectively) were similar to their substrate Km values. The kinetic properties of this enzyme, as well as estimates of the cellular levels of its potential substrates, indicate that inositol pentakisphosphate and inositol hexakisphosphate are likely to be the preferred substrates in vivo. << Less

  J. Biol. Chem. 266:16499-16506(1991) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.