An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called ‘high-energy’ phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.

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Tumour cells can employ extracellular Ins(1,2,3,4,5,6)P6 and multiple inositol-polyphosphate phosphatase 1 (MINPP1) dephosphorylation to improve their proliferation

Biochemical Journal ◽

10.1042/bj20121524 ◽

2013 ◽

Vol 450 (1) ◽

pp. 115-125 ◽

Cited By ~ 35

Author(s):

Sabine Windhorst ◽

Hongying Lin ◽

Christine Blechner ◽

Werner Fanick ◽

Laura Brandt ◽

...

Keyword(s):

Mammalian Cells ◽

Inositol Phosphate ◽

Tumour Cells ◽

Growth Potential ◽

Metal Phosphate ◽

Dependent Manner ◽

Inositol Polyphosphate ◽

Surface Metal ◽

Rna Knockdown ◽

Tumour Cell Growth

InsP6 [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 μM. We noticed that InsP6 in culture medium at a concentration of ≤50 μM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 μM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal–InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.

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Novel Substrates for Kinases Involved in the Biosynthesis of Inositol Pyrophosphates and Their Enhancement of ATPase Activity of a Kinase

Molecules ◽

10.3390/molecules26123601 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3601

Author(s):

Raja Mohanrao ◽

Ruth Manorama ◽

Shubhra Ganguli ◽

Mithun C. Madhusudhanan ◽

Rashna Bhandari ◽

...

Keyword(s):

Enzyme Activity ◽

Atpase Activity ◽

Catalytic Domain ◽

Inositol Phosphate ◽

Substrate Recognition ◽

Competitive Inhibitor ◽

Inositol Polyphosphates ◽

Inositol Pyrophosphate ◽

Phosphate Donor ◽

Inositol Pyrophosphates

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo-inositol polyphosphates—scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5—from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

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Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells

Biochemical Journal ◽

10.1042/bj3060557 ◽

1995 ◽

Vol 306 (2) ◽

pp. 557-564 ◽

Cited By ~ 21

Author(s):

C J Barker ◽

P J French ◽

A J Moore ◽

T Nilsson ◽

P O Berggren ◽

...

Keyword(s):

Beta Cells ◽

Mammalian Cells ◽

Lymphoid Cells ◽

Inositol Polyphosphate ◽

Monocytic Differentiation ◽

Isotopic Equilibrium ◽

Inositol Polyphosphates ◽

Hl60 Cells ◽

Cellular Iron ◽

In Cells

1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.

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Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

Infection and Immunity ◽

10.1128/iai.01421-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1245-1255 ◽

Cited By ~ 25

Author(s):

Sophie Lev ◽

Desmarini Desmarini ◽

Cecilia Li ◽

Methee Chayakulkeeree ◽

Ana Traven ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Phospholipase C ◽

Mammalian Cells ◽

Galleria Mellonella ◽

Inositol Trisphosphate ◽

Infection Model ◽

Inositol Polyphosphate ◽

Inositol Polyphosphates ◽

Content Type ◽

Capsule Production

ABSTRACTPhospholipase C (PLC) ofCryptococcus neoformans(CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP2) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP3). InSaccharomyces cerevisiae, Plc1-derived IP3is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3to more complex inositol polyphosphates. In this study, we show that inC. neoformans, the enzyme encoded byARG1is the major IP3kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in theGalleria mellonellainfection model. IP3content was reduced in the CnΔplc1mutant and markedly increased in the CnΔarg1mutant, while PIP2was increased in both mutants. The CnΔplc1and CnΔarg1mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to theS. cerevisiae ARG82deletion mutant (ScΔarg82) strain, the CnΔarg1mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3causes Ca2+release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence ofC. neoformansand further show that production of IP3as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.

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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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Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalks between SA-dependent immunity and phosphate-starvation responses

10.1101/2021.01.27.428180 ◽

2021 ◽

Author(s):

Hitika Gulabani ◽

Krishnendu Goswami ◽

Yashika Walia ◽

Jewel Jameeta Noor ◽

Kishor D. Ingole ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Phosphate Starvation ◽

High Energy ◽

Signaling Networks ◽

Inositol Polyphosphate ◽

Gene Expressions ◽

Inositol Polyphosphates ◽

A Cell ◽

The Common ◽

Cellular Pathways

AbstractThe propensity for polyphosphorylation makes myo-inositol derivatives, the inositol polyphosphates (InsPs), especially phytic acid or inositol hexakisphosphate (InsP6) the major form of phosphate storage in plants. Acts of pyrophosphorylation on InsP6 generates InsP7 or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. Also implicated as co-factors for several phytohormone signaling networks, InsP7/InsP8 modulate key developmental processes. With recent identification as the common moeity for transducing both jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 is the classic example of a metabolite that may moonlight crosstalks to different cellular pathways during diverse stress adaptations. We show here that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2), but not other InsP-kinases, suppress basal salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, elevated endogenous SA levels and constitutive activation of defense signaling lead to enhanced resistance against the virulent Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that activated SA-signaling sectors in these mutants modulate expression amplitudes of phosphate-starvation inducible (PSI)-genes, reported earlier. In turn, via mutualism the heightened basal defenses in these mutants require upregulated PSI-gene expressions likely highlighting the increased demand of phosphates required to support immunity. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators among diverse signaling networks programming stress-appropriate adaptations.

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A pyro-phosphodegron controls MYC polyubiquitination to regulate cell survival

10.1101/2020.02.12.945634 ◽

2020 ◽

Author(s):

Padmavathi Lolla ◽

Akruti Shah ◽

C.P. Unnikannan ◽

Vineesha Oddi ◽

Rashna Bhandari

Keyword(s):

Cell Survival ◽

Posttranslational Modification ◽

High Energy ◽

Growth Stress ◽

Environmental Cues ◽

Dependent Manner ◽

Inositol Pyrophosphate ◽

Inositol Pyrophosphates ◽

Survival And Growth ◽

Insight Into

ABSTRACTThe transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. Here, we report that serine pyrophosphorylation – an enigmatic posttranslational modification triggered by inositol pyrophosphate signaling molecules – controls MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Pyrophosphorylation of MYC promotes its interaction with the E3 ubiquitin ligase FBW7, thereby enhancing MYC polyubiquitination and degradation. FBW7 can bind directly to the PEST domain of MYC in a pyrophosphorylation-dependent manner. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells, and promotes cell proliferation in response to mitogens. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

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Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis

BMC Biology ◽

10.1186/s12915-021-01198-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Anuj Shukla ◽

Mandeep Kaur ◽

Swati Kanwar ◽

Gazaldeep Kaur ◽

Shivani Sharma ◽

...

Keyword(s):

Cell Wall ◽

Drought Tolerance ◽

Inositol Phosphate ◽

High Energy ◽

Variable Number ◽

Future Research ◽

Water Deficit Stress ◽

Transcriptional Level ◽

Inositol Pyrophosphate ◽

Transgenic Lines

Abstract Background Inositol pyrophosphates (PP-InsPs) are high-energy derivatives of inositol, involved in different signalling and regulatory responses of eukaryotic cells. Distinct PP-InsPs species are characterized by the presence of phosphate at a variable number of the 6-carbon inositol ring backbone, and two distinct classes of inositol phosphate kinases responsible for their synthesis have been identified in Arabidopsis, namely ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were previously shown to control defense against pathogens and phosphate response signals. However, other potential roles of plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. Results Here, we characterized the physiological functions of two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2. We demonstrate that wheat VIH proteins can utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Ectopic overexpression of TaVIH2-3B in Arabidopsis confers tolerance to drought stress and rescues the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B-expressing transgenic lines of Arabidopsis shows genome-wide reprogramming with remarkable effects on genes involved in cell-wall biosynthesis, which is supported by the observation of enhanced accumulation of polysaccharides (arabinogalactan, cellulose, and arabinoxylan) in the transgenic plants. Conclusions Overall, this work identifies a novel function of VIH proteins, implicating them in modulation of the expression of cell-wall homeostasis genes, and tolerance to water-deficit stress. This work suggests that plant VIH enzymes may be linked to drought tolerance and opens up the possibility of future research into using plant VIH-derived products to generate drought-resistant plants.

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Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011376 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9366-9378 ◽

Cited By ~ 7

Author(s):

Uriel López-Sánchez ◽

Sandrine Tury ◽

Gaël Nicolas ◽

Miranda S. Wilson ◽

Snejana Jurici ◽

...

Keyword(s):

Phosphate Uptake ◽

Neuropsychiatric Symptoms ◽

Binding Pocket ◽

Inositol Polyphosphate ◽

Phosphate Homeostasis ◽

Inositol Polyphosphates ◽

Inositol Pyrophosphate ◽

Atp Levels ◽

Primary Familial Brain Calcification ◽

Brain Calcification

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP–binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

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Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651597 ◽

1993 ◽

Vol 69 (03) ◽

pp. 286-292 ◽

Cited By ~ 13

Author(s):

Che-Ming Teng ◽

Feng-Nien Ko ◽

Inn-Ho Tsai ◽

Man-Ling Hung ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Inositol Phosphate ◽

Shape Change ◽

Platelet Activating Factor ◽

Atp Release ◽

Platelet Membrane ◽

Thromboxane B2 ◽

Dependent Manner ◽

Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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