Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.
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ABSTRACT:
SUBMITTER: Moure MJ
PROVIDER: S-EPMC5690875 | biostudies-literature | 2017 Nov
REPOSITORIES: biostudies-literature
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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]
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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.
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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).
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2021-10-29 | PXD026653 | Pride