Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.
Ontology highlight
ABSTRACT:
SUBMITTER: Moure MJ
PROVIDER: S-EPMC5690875 | biostudies-literature | 2017 Nov
REPOSITORIES: biostudies-literature
Publications
Perdeuterated and <sup>13</sup>C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.
Moure M J MJ Zhuo Y Y Boons G J GJ Prestegard J H JH
Chemical communications (Cambridge, England) 20171101 92
The synthesis of perdeuterated and <sup>13</sup>C enriched myo-inositol is presented. Myo-inositol and its derivatives are of interest as substrates for enzymes producing phosphorylated species with regulatory functions in many organisms. Its utility in monitoring real-time phosphorylation by myo-inositol-3-kinase is illustrated using dynamic nuclear polarization (DNP) to enhance NMR observation. ...[more]
Similar Datasets
Project description:myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using (32)Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS.
| S-EPMC3772229 | biostudies-literature
Project description:1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate, 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346, 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).
| S-EPMC1150078 | biostudies-other
Project description:BACKGROUNDMyo-inositol (MI), successfully used in polycystic ovary syndrome (PCOS), was administered with ?-LA to exploit its action of favouring the passage of other molecules through biological barriers, and also considering its anti-inflammatory effect.METHODSPCOS patients, according to the Rotterdam ESHRE-ASRM criteria, with anovulation and infertility >?1 year, were included in this open and prospective study. The preliminary phase was aimed at determining a set of MI-resistant PCOS patients. This treatment involved 2 g MI, taken twice per day by oral route, for three months. The Homeostasis Model Assessment (HOMA) index and MI plasma levels were measured. In the main phase, previously selected MI-resistant patients received the same daily amount of MI plus 50 mg ?-LA twice a day, for a further three months. Ovulation was assessed using ultrasound examination on days 12, 14 and 20 of the cycle. The HOMA index, lipid, hormone and MI plasma levels were detected at baseline and at the end of this phase.RESULTSThirty-seven anovulatory PCOS subjects were included in the study. Following MI treatment, 23 of the 37 women (62%) ovulated, while 14 (38%) were resistant and did not ovulate. In the latter group, MI plasma levels did not increase. These MI-resistant patients underwent treatment in the main phase of the study, receiving MI and ?-LA. After this combined treatment, 12 (86%) of them ovulated. Their MI plasma levels were found to be significantly higher than at baseline; also, a hormone and lipid profile improvement was recorded.CONCLUSIONThe combination of MI with ?-LA allowed us to obtain significant progress in the treatment of PCOS MI-resistant patients. Therefore, this new formulation was able to re-establish ovulation, greatly increasing the chances of desired pregnancy.TRIAL REGISTRATIONClinical trial registration number: NCT03422289 ( ClinicalTrials.gov registry).
| S-EPMC5944130 | biostudies-literature
Project description:Phytate forms insoluble precipitates with various cations that are recalcitrant to digestion in poultry. Dietary supplementation with exogenous phytase has been shown to improve phytate solubility and digestibility and, in turn, improve animal growth performance. Although the kinetics of phytate hydrolysis by exogenous phytase are well described in vitro, the progression of the reaction in vivo is still not well defined. The aim of the present study was, therefore, to monitor the kinetic variation of myo-inositol (myo-Ins) levels in both circulation and feather following exogenous phytase supplementation. In experiment 1, 4 week-old male broilers were individually housed with ad libitum access to water and a standard commercial diet. Birds were maintained under environmental temperature of 24°C and 30% RH. Birds were cannulated in the cutaneous ulnar vein on the right wing and remained untouched for 3 days. On the day of the experiment, birds were randomly divided into three body weight-matched groups and fed either the control diet, the control diet-supplemented with myo-Ins or Ronozyme HiPhos (0.06%, DSM Nutritional Products, Switzerland) for 10 h. In the experiment 2, birds were fed only HiPhos for 30 h. Growing feathers and blood were collected at baseline and then every 2 h for 10 h (experiment 1) and 30 h (experiment 2) post-prandially. Plasma and feather myo-Ins levels were determined by UHPLC-MS/MS. The relative expression of inositol polyphosphate-1-phosphatase (INPP1), inositol hexakisphosphate kinase 1-3 (IP6K1-3), inositol-3-phosphate synthase (ISYNA), and multiple inositol-polyphosphate phosphatase 1 (MNPP1) genes in blood and feathers was determined by real-time qPCR using 2-??Ct method. Plasma and feather myo-Ins levels were significantly increased by HiPhos at 6 h to 8 h post-prandial. The mRNA abundances of INPP1, IP6K1, and ISYNA in the circulation were significantly down regulated at all periods compared to the baseline levels. IP6K2, IP6K3, and MINPP1 gene expression, however, was up regulated at 8 h post-prandial and then returned to the baseline levels. In feathers, the expression of INPP1 was induced at 8 h post-prandial and remained higher compared to the baseline. The expression of IP6K2, IP6K3, and MINPP1 was down regulated during the first 10 h and then returned to baseline levels for the rest of the post-prandial period. Taken together, our data show that phytase modulates the expression of genes associated with myo-Ins metabolism and generates release of myo-Ins in both circulation and feather at 6-10 h post-feeding. Feather myo-Ins concentration could be used as a non-invasive method to monitor phytate hydrolysis in practice.
| S-EPMC7333251 | biostudies-literature
Project description:AIM:To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate p?-cateninS552 as a biomarker of recurrent dysplasia. METHODS:We examined the effects of dietary myo-inositol treatment on inflammation, p?-cateninS552 and pAkt levels by histology and western blot in IL-10-/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear p?-cateninS552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia. RESULTS:In mice, p?-cateninS552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, p?-cateninS552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease. CONCLUSION:Enumerating crypts with increased numbers of p?-cateninS552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials.
| S-EPMC5537179 | biostudies-literature
Project description:E. histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although the set of proteins (peroxins) responsible for peroxisome biogenesis was reduced to only seven members (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0,044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living M. balamuthi and P. schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
2021-10-29 | PXD026653 | Pride
Project description:Multinuclear n.m.r. spectroscopy has been used to confirm the identify of inositol 1,4,5-trisphosphate and to deduce its molecular conformation as having five equatorial and one axial ring substituents.
| S-EPMC1153016 | biostudies-other
Project description:The enzyme myo-inositol oxygenase is the key enzyme of a pathway leading from myo-inositol to UDP-glucuronic acid. In Arabidopsis, myo-inositol oxygenase is encoded by four genes. All genes are strongly expressed in syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots. Here, we studied the effect of a quadruple myo-inositol oxygenase mutant on nematode development. We performed metabolite profiling of syncytia induced in roots of the myo-inositol oxygenase quadruple mutant. The role of galactinol in syncytia was studied using Arabidopsis lines with elevated galactinol levels and by supplying galactinol to wild-type seedlings. The quadruple myo-inositol oxygenase mutant showed a significant reduction in susceptibility to H. schachtii, and syncytia had elevated myo-inositol and galactinol levels and an elevated expression level of the antimicrobial thionin gene Thi2.1. This reduction in susceptibility could also be achieved by exogenous application of galactinol to wild-type seedlings. The primary function of myo-inositol oxygenase for syncytium development is probably not the production of UDP-glucuronic acid as a precursor for cell wall polysaccharides, but the reduction of myo-inositol levels and thereby a reduction in the galactinol level to avoid the induction of defence-related genes.
| S-EPMC4285123 | biostudies-literature
Project description:Myo-inositol is a ubiquitous metabolite of plants. It is synthesized by a highly conserved enzyme L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4). Myo-inositol is well characterized during abiotic stress tolerance but its role during growth and development is unclear. In this study, we demonstrate that the apical hook maintenance and hypocotyl growth depend on myo-inositol. We discovered the myo-inositol role during hook formation and its maintenance via ethylene pathway in Arabidopsis by supplementation assays and qPCR. Our results suggest an essential requirement of myo-inositol for mediating the ethylene response and its interaction with brassinosteroid to regulate the skotomorphogenesis. A model is proposed outlining how MIPS regulates apical hook formation and hypocotyl growth.
| S-EPMC7567114 | biostudies-literature
Project description:Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) as the sole carbon and energy source is characterized by a bistable phenotype that manifests in a growth phenotype with an extraordinarily long and length-variable lag phase. However, in the presence of hydrogen carbonate, in the absence of IolR that represses the MI degradation pathway, or if cells are already adapted to minimal medium (MM) with MI, the lag phase is drastically shortened, and the bistable phenotype is abolished. We hypothesized that memory development or hysteresis is a further characteristic of MI degradation by S. Typhimurium; therefore, we investigated the transition from a short to a long lag phase in more detail. Growth experiments demonstrated that memory on the population level is successively lost within approximately 8 hr after cells, which had been adapted to MI utilization, were transferred to lysogeny broth (LB) medium. Flow cytometry (FC) analysis using a chromosomal fusion to PiolE , a promoter controlling the expression of the enzymatic genes iolE and iolG involved in MI degradation, indicated a gradual reversion within a few hours from a population in the "ON" status with respect to iolE transcription to one that is mainly in the "OFF" status. Growth and FC experiments revealed that IolR does not affect hysteresis.
| S-EPMC5387303 | biostudies-literature