Metabolic Labeling of Inositol Phosphates and Phosphatidylinositols in Yeast and Mammalian Cells

High-resolution imaging of choline metabolites in living mammalian cells, primary neurons andC. eleganshas been demonstrated with the potential forin vivodisease detection and developmental monitoring.

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The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

Biochemical Journal ◽

10.1042/bj2640323 ◽

1989 ◽

Vol 264 (2) ◽

pp. 323-333 ◽

Cited By ~ 25

Author(s):

T Radenberg ◽

P Scholz ◽

G Bergmann ◽

G W Mayr

Keyword(s):

Mammalian Cells ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Detection System ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Phosphate Metabolism ◽

Qualitative And Quantitative ◽

Inositol Phosphate Metabolism ◽

Avian Erythrocytes

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

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A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

10.1101/2021.09.01.458261 ◽

2021 ◽

Author(s):

Thomas E. Wood ◽

Jessica M. Yoon ◽

Heather D. Eshleman ◽

Daniel J. Slade ◽

Cammie F. Lesser ◽

...

Keyword(s):

Mammalian Cells ◽

Inositol Phosphates ◽

Bacterial Pathogen ◽

Cysteine Proteases ◽

Effector Proteins ◽

Host Cells ◽

Expression Library ◽

Inositol Pyrophosphates ◽

Type 3

Shigella spp. cause diarrhea by invading human intestinal epithelial cells. Effector proteins delivered into target host cells by the Shigella type 3 secretion system modulate host signaling pathways and processes in a manner that promotes infection. The effector OspB activates mTOR, the central cellular regulator of growth and metabolism, and potentiates the inhibition of mTOR by rapamycin. The net effect of OspB on cell monolayers is cell proliferation at infectious foci. To gain insights into the mechanism by which OspB potentiates rapamycin inhibition of mTOR, we employ in silico analyses to identify putative catalytic residues of OspB and show that a conserved cysteine-histidine dyad is required for this activity of OspB. In a screen of an over-expression library in Saccharomyces cerevisiae, we identify a dependency of OspB activity on inositol pyrophosphates, a class of eukaryotic secondary messengers that are distinct from the inositol phosphates known to act as cofactors for bacterial cysteine proteases. We show that inositol pyrophosphates are required for OspB activity not only in yeast, but also in mammalian cells - the first demonstration of inositol pyrophosphates being required for virulence of a bacterial pathogen in vivo.

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Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

Nature Biotechnology ◽

10.1038/nbt.1861 ◽

2011 ◽

Vol 29 (5) ◽

pp. 436-442 ◽

Cited By ~ 348

Author(s):

Michal Rabani ◽

Joshua Z Levin ◽

Lin Fan ◽

Xian Adiconis ◽

Raktima Raychowdhury ◽

...

Keyword(s):

Mammalian Cells ◽

Metabolic Labeling

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Role of Inositols and Inositol Phosphates in Energy Metabolism

Molecules ◽

10.3390/molecules25215079 ◽

2020 ◽

Vol 25 (21) ◽

pp. 5079 ◽

Cited By ~ 2

Author(s):

Saimai Chatree ◽

Nanthaphop Thongmaen ◽

Kwanchanit Tantivejkul ◽

Chantacha Sitticharoon ◽

Ivana Vucenik

Keyword(s):

Insulin Resistance ◽

Insulin Sensitivity ◽

Energy Metabolism ◽

Mammalian Cells ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Biological Activities ◽

High Energy ◽

Beneficial Effects ◽

Treatment And Prevention

Recently, inositols, especially myo-inositol and inositol hexakisphosphate, also known as phytic acid or IP6, with their biological activities received much attention for their role in multiple health beneficial effects. Although their roles in cancer treatment and prevention have been extensively reported, interestingly, they may also have distinctive properties in energy metabolism and metabolic disorders. We review inositols and inositol phosphate metabolism in mammalian cells to establish their biological activities and highlight their potential roles in energy metabolism. These molecules are known to decrease insulin resistance, increase insulin sensitivity, and have diverse properties with importance from cell signaling to metabolism. Evidence showed that inositol phosphates might enhance the browning of white adipocytes and directly improve insulin sensitivity through adipocytes. In addition, inositol pyrophosphates containing high-energy phosphate bonds are considered in increasing cellular energetics. Despite all recent advances, many aspects of the bioactivity of inositol phosphates are still not clear, especially their effects on insulin resistance and alteration of metabolism, so more research is needed.

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ITPK1 mediates the lipid-independent synthesis of inositol phosphates controlled by metabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911431116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24551-24561 ◽

Cited By ~ 18

Author(s):

Yann Desfougères ◽

Miranda S. C. Wilson ◽

Debabrata Laha ◽

Gregory J. Miller ◽

Adolfo Saiardi

Keyword(s):

Phospholipase C ◽

Mammalian Cells ◽

Inositol Phosphates ◽

Phosphate Starvation ◽

Dependent Manner ◽

Social Amoeba ◽

Functional Roles ◽

Inositol Tetrakisphosphate ◽

Independent Synthesis ◽

Multiple Signaling

Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP3 generated from PIP2 by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative “soluble” route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)—found in Asgard archaea, social amoeba, plants, and animals—phosphorylates I(3)P1 originating from glucose-6-phosphate, and I(1)P1 generated from sphingolipids, to enable synthesis of IP6. We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP6 levels in a ITPK1-dependent manner, establishing a route to IP6 controlled by cellular metabolic status, that is not detectable by traditional [3H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote.

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A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

Open Biology ◽

10.1098/rsob.150014 ◽

2015 ◽

Vol 5 (3) ◽

pp. 150014 ◽

Cited By ~ 71

Author(s):

Miranda S. C. Wilson ◽

Simon J. Bulley ◽

Francesca Pisani ◽

Robin F. Irvine ◽

Adolfo Saiardi

Keyword(s):

Human Plasma ◽

Mammalian Cells ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Genetic Studies ◽

Signalling Molecules ◽

Tissue Extracts ◽

Mammalian Cell Lines ◽

Novel Method ◽

Purification Protocol

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO 2 ) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP 6 (phytate), its pyrophosphate derivatives InsP 7 and InsP 8 , and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP 6 , InsP 7 and InsP 8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO 2 bead purification also enabled us to quantify InsP 6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP 6 phosphatase in human plasma, and secondly, InsP 6 is undetectable in either fluid. These observations seriously question reports that InsP 6 is present in human biofluids and the advisability of using InsP 6 as a dietary supplement.

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