Search

Inositol phosphates (IPs) are phosphorylated derivatives of myo-inositol involved in the regulation of several cellular processes through their interaction with specific proteins. Their synthesis relies on the activity of specific kinases that use ATP as phosphate donor. Here, we combined reverse genetics and liquid chromatography coupled to mass spectrometry (LC-MS) to dissect the inositol phosphate biosynthetic pathway and its metabolic intermediates in the main life cycle stages (epimastigotes, cell-derived trypomastigotes, and amastigotes) of Trypanosoma cruzi, the etiologic agent of Chagas disease. We found evidence of the presence of highly phosphorylated IPs, like inositol hexakisphosphate (IP6), inositol heptakisphosphate (IP7), and inositol octakisphosphate (IP8), that were not detected before by HPLC analyses of the products of radiolabeled exogenous inositol. The kinases involved in their synthesis (inositol polyphosphate multikinase (TcIPMK), inositol 5-phosphate kinase (TcIP5K), and inositol 6-phosphate kinase (TcIP6K)) were also identified. TcIPMK is dispensable in epimastigotes, important for the synthesis of polyphosphate, and critical for the virulence of the infective stages. TcIP5K is essential for normal epimastigote growth, while TcIP6K mutants displayed defects in epimastigote motility and growth. Our results demonstrate the relevance of highly phosphorylated IPs in the life cycle of T. cruzi.

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living β-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts β-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP(8)). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP(8) synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP(8)) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP(8) levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP(8); PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP(8) to the XPR1 N-terminus (K(d) = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. As 18O can be derived from heavy water (H218O), it is comparably cheap and particularly suited for labelling of phosphorylated compounds, provided the introduction is straight-forward and phosphate neutral loss in the ion source can be avoided. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2‑phosphoramidite reagents. This flexible toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including - but not limited to - nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18O-enrichment ratios >95% and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18O labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices, such as mammalian cell lysates, slime mold and plant samples. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionization triple quadrupole mass spectrometry.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

Inositol pyrophosphates (PP-InsPs), including diphospho-myo-inositol pentakisphosphate (5-InsP7) and bis-diphospho-myo-inositol tetrakisphosphate (1,5-InsP8), are highly polar, membrane-impermeant signaling molecules that control many homeostatic responses to metabolic and bioenergetic imbalance. To delineate their molecular activities, there is an increasing need for a toolbox of methodologies for real-time modulation of PP-InsP levels inside large populations of cultured cells. Here, we describe procedures to package PP-InsPs into thermosensitive phospholipid nanocapsules that are impregnated with a near infra-red photothermal dye; these liposomes are readily accumulated into cultured cells. The PP-InsPs remain trapped inside the liposomes until the cultures are illuminated with a near infra-red light-emitting diode (LED) which permeabilizes the liposomes to promote PP-InsP release. Additionally, so as to optimize these procedures, a novel stably fluorescent 5-InsP7 analogue (i.e., 5-FAM-InsP7) was synthesized with the assistance of click-chemistry; the delivery and deposition of the analogue inside cells was monitored by flow cytometry and by confocal microscopy. We describe quantitatively-controlled PP-InsP release inside cells within 5 min of LED irradiation, without measurable effect upon cell integrity, using a collimated 22 mm beam that can irradiate up to 106 cultured cells. Finally, to interrogate the biological value of these procedures, we delivered 1,5-InsP8 into HCT116 cells and showed it to dose-dependently stimulate the rate of [33P]-Pi uptake; these observations reveal a rheostatic range of concentrations over which 1,5-InsP8 is biologically functional in Pi homeostasis.

The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP8, and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP8. Furthermore, we showed that InsP8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.

Diphospho-myo-inositol polyphosphates, also termed inositol pyrophosphates, are molecular messengers containing at least one high-energy phosphoanhydride bond and regulate a wide range of cellular processes in eukaryotes. While inositol pyrophosphates InsP7 and InsP8 are present in different plant species, both the identity of enzymes responsible for InsP7 synthesis and the isomer identity of plant InsP7 remain unknown. This study demonstrates that Arabidopsis ITPK1 and ITPK2 catalyze the phosphorylation of phytic acid (InsP6) to the symmetric InsP7 isomer 5-InsP7 and that the InsP6 kinase activity of ITPK enzymes is evolutionarily conserved from humans to plants. We also show by 31P nuclear magnetic resonance that plant InsP7 is structurally identical to the in vitro InsP6 kinase products of ITPK1 and ITPK2. Our findings lay the biochemical and genetic basis for uncovering physiological processes regulated by 5-InsP7 in plants.

Phosphate's central role in most biochemical reactions in a living organism requires carefully maintained homeostasis. Although phosphate homeostasis in mammals has long been studied at the organismal level, the intracellular mechanisms controlling phosphate metabolism are not well-understood. Inositol pyrophosphates have emerged as important regulatory elements controlling yeast phosphate homeostasis. To verify whether inositol pyrophosphates also regulate mammalian cellular phosphate homeostasis, here we knocked out inositol hexakisphosphate kinase (IP6K) 1 and IP6K2 to generate human HCT116 cells devoid of any inositol pyrophosphates. Using PAGE and HPLC analysis, we observed that the IP6K1/2-knockout cells have nondetectable levels of the IP6-derived IP7 and IP8 and also exhibit reduced synthesis of the IP5-derived PP-IP4. Nucleotide analysis showed that the knockout cells contain increased amounts of ATP, whereas the Malachite green assay found elevated levels of free intracellular phosphate. Furthermore, [32Pi] pulse labeling experiments uncovered alterations in phosphate flux, with both import and export of phosphate being decreased in the knockout cells. Functional analysis of the phosphate exporter xenotropic and polytropic retrovirus receptor 1 (XPR1) revealed that it is regulated by inositol pyrophosphates, which can bind to its SPX domain. We conclude that IP6K1 and -2 together control inositol pyrophosphate metabolism and thereby physiologically regulate phosphate export and other aspects of mammalian cellular phosphate homeostasis.

T cell-independent (TI) B cell response is critical for the early protection against pathogen invasion. The regulation and activation of Bruton’s tyrosine kinase (Btk) is known as a pivotal step of B cell antigen receptor (BCR) signaling in TI humoral immunity, as observed in patients with X-linked agammaglobulinemia (XLA) experiencing a high incidence of encapsulated bacterial infections. However, key questions remain as to whether a well-established canonical BCR signaling pathway is sufficient to regulate the activity of Btk. Here, we find that inositol hexakisphosphate (InsP(6)) acts as a physiological regulator of Btk in BCR signaling. Absence of higher order inositol phosphates (InsPs), inositol polyphosphates, leads to an inability to mount immune response against TI antigens. Interestingly, the significance of InsP(6)-mediated Btk regulation is more prominent in IgM(+) plasma cells. Hence, the present study identifies higher order InsPs as principal components of B cell activation upon TI antigen stimulation and presents a mechanism for InsP-mediated regulation of the BCR signaling.

5-InsP7 is an endogenous negative regulator of Na+/K+-ATPase., Sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) is one of the most abundant cell membrane proteins and is essential for eukaryotes. Endogenous negative regulators have long been postulated to play an important role in regulating the activity and stability of Na+/K+-ATPase, but characterization of these regulators has been elusive. Mechanisms of regulating Na+/K+-ATPase homeostatic turnover are unknown. Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7), generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream degradation of Na+/K+-ATPase-α1. Deletion of IP6K1 elicits a twofold enrichment of Na+/K+-ATPase-α1 in plasma membranes of multiple tissues and cell types. Using a suite of synthetic chemical biology tools, we found that 5-InsP7 binds the RhoGAP domain of phosphatidylinositol 3-kinase (PI3K) p85α to disinhibit its interaction with Na+/K+-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers the clathrin-mediated endocytosis of Na+/K+-ATPase-α1. Our study identifies 5-InsP7 as an endogenous negative regulator of Na+/K+-ATPase-α1.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of Pi starvation response transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP7 from InsP6in vitro, but the importance of this activity in Pi signaling remained unknown. Here, we show that uncontrolled Pi accumulation in ITPK1-deficient plants is accompanied by impaired Pi-dependent InsP7 and InsP8 synthesis. Reciprocal grafting demonstrates that Pi starvation responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance assays and PAGE analyses with recombinant protein reveal that besides InsP6 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP7 but not any other InsP7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes in InsP7 and InsP8 to cellular Pi are conserved from land plant species to human cells, suggesting that Pi-dependent PP-InsP synthesis is a common component of Pi signaling across kingdoms. Together, our study demonstrates how Pi-dependent changes in nutritional and energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.

β-Cells respond directly to the intracellular photochemical release of caged inositol pyrophosphate isomers with modulations of oscillations in cytosolic Ca2+., Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases (IP6Ks, PPIP5Ks). These approaches are, inter alia, limited by time-resolution and potential compensation mechanisms. Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP5, the major cellular inositol pyrophosphate, oscillations of intracellular Ca2+ levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP5, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca2+]i oscillations, a key element in triggering exocytosis and secretion in β-cells.

The combinatorial phosphorylation ofmyo-inositolresults in the generation of different inositol phosphates (InsP), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6is also the precursor of higher phosphorylated forms called inositol pyrophosphates (PP-InsPs), such as InsP7and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6kinases. Recently, it was shown that Arabidopsis ITPK1 catalyzes the phosphorylation of InsP6to the natural 5-InsP7isomerin vitro. Here, we demonstrate that Arabidopsis ITPK1 contributes to the synthesis of InsP7in planta. We further find a critical role of ITPK1 in auxin-related processes including primary root elongation, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity towards exogenously applied auxin. Notably, 5-InsP7binds to recombinant auxin receptor complex, consisting of the F-Box protein TIR1, ASK1 and the transcriptional repressor IAA7, with high affinity. Furthermore, a specific increase in 5-InsP7in a heterologous yeast expression system results in elevated interaction of the TIR1 homologs AFB1 and AFB2 with various AUX/IAA-type transcriptional repressors. We also identified a physical interaction between ITPK1 and TIR1, suggesting a dedicated channeling of an activating factor, such as 5-InsP7, to the auxin receptor complex. Our findings expand the mechanistic understanding of auxin perception and lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP7.

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.

The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.

The free energy of nucleotide hydrolysis depends on phosphate concentration. Cells regulate cytosolic phosphate levels by orchestrating phosphate acquisition and storage through inositol pyrophosphates (PP-InsP) and SPX domains. Here, we report the synthesis of the novel 5-PPP-InsP5 containing a triphosphate subunit. Using this and a series of synthetic PP-InsP, we examined the ligand specificity of the SPX domain in the PP-InsP-controlled yeast polyphosphate polymerase VTC. SPX decodes the relative positioning of the phosphoric anhydrides, their structure (diphosphate vs triphosphate), and the presence of other phosphates on the inositol ring. Despite the higher potency of 1,5-(PP)2-InsP4, 5-PP-InsP5 is the primary activator of VTC in cells, indicating that its higher concentration compensates for its lower potency. 1,5-(PP)2-InsP4 levels rise and could become relevant under stress conditions. Thus, SPX domains may integrate PP-InsP dependent signaling to adapt cytosolic phosphate concentrations to diffe...

The specific non-invasive control of intracellular signaling events requires advanced tools that enter cells by diffusion and are controllable by light. Here, we detail the synthesis and application of membrane-permeant caged inositol pyrophosphates with respect to cell entry and cell distribution. We recently published the synthesis of these tools as well as their effect on PH-domain localization in HeLa cells and oscillations of the intracellular calcium concentration in β-cells, which are known to drive insulin secretion. In this chapter, we discuss the possibilities and limitations when using cell-penetrating inositol pyrophosphates. We provide a detailed protocol for the application in live mouse β-cells and we discuss the image analysis needed for following effects on calcium signaling.

Inositol pyrophosphates (PP-InsPs) are “energetic” intracellular signals that are ubiquitous in animals, plants, and fungi. We are characterizing new proteins that regulate PP-InsP metabolism and/o...

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains. We have previously shown that these domains act as cellular receptors for inositol pyrophosphate (PP-InsP) signaling molecules, suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that simultaneous deletion of two diphosphoinositol pentakisphosphate kinases VIH1 and 2 in Arabidopsis impairs plant growth and leads to constitutive Pi starvation responses. We demonstrate that VIH1 and VIH2 are bifunctional cytosolic enzymes able to generate and break-down PP-InsPs. Point-mutants targeting the kinase and phosphatase active sites have opposing effects on plant Pi content and Pi starvation responses, while VIH1 and VIH2 protein levels remain constant in different Pi growth conditions. Enzymatic assays reveal that ATP-Mg2+ substrate levels can shift the relative kinase and phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases. Deletion of phosphate starvation response transcription factors rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases and PP-InsPs in plant phosphate signal transduction cascades. We propose that VIH1 and VIH2 relay changes in cellular ATP concentration to changes in PPInsP levels, allowing plants to maintain cellular Pi concentrations constant and to trigger Pi starvation responses.

The propagation of information inside cells is achieved in nature by small diffusible molecules. Many of these secondary messengers are highly charged species derived from myo -inositol. The diphosphoinositol polyphosphates (X-PP- myo -InsP 5 ) represent a novel group of secondary messengers that regulate various important biological processes. Depending on the phosphorylation pattern, these molecules can occur in symmetric or nonsymmetric forms. In a recent study, we have shown the potential of C 2 symmetric phosphoramidites as reagents for desymmetrizing myo -inositol and for obtaining all four possible nonsymmetric X-PP- myo -InsP 5 stereoisomers in enantiomerically pure form. These results and their potential implications for the design of tools for chemical biology are discussed.

Highly charged and unstable diphosphoinositol pentaphosphates (PP InsP5) are challenging but biologically relevant synthetic targets. A novel C2 symmetric phosphoramidite was developed that can be used to target all 4 unsymmetric PP InsP5. It does not only serve well in the desymmetrization but is also an excellent phosphate protecting group. With this approach the target structures were synthesized in few steps and high enantiomeric ratios. With the obtained compounds specificity of Ddp1 from yeast was studied.

Desymmetrization by phosphorylation represents a promising method with potential impact in many different areas of research. C2-Symmetric phosphoramidites have been used to desymmetrize myo-inositol derivatives by functionalization at different positions. With this method, 1:1 mixtures of diastereomers are obtained that can be separated subsequently. In this work, activation of a C2-symmetric phosphoramidite is achieved by addition of pentafluorophenol (PFP) and leads to a reactive PFP phosphite, which can then be coupled to protected myo-inositol derivatives with reactive OH groups at the 1, 3, 4 and 6 positions. This strategy enhances the diastereoselectivity of the coupling reaction with a preference towards phosphitylation at position 6 (up to 3:1) or position 3 (up to 2:1). The concept of attenuative activation of phosphoramidites via in situ generated pentafluorophenol phosphite triesters is thus proven in these studies. It is further shown that Lewis-Acid catalysis enhances the rate of phosphite triester coupling without affecting the diastereoselectivity. This novel strategy improves access to different phosphorylated myo-inositol derivatives and will thus enable further studies into the function of these important intracellular second messengers.

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Diphosphoinositol polyphosphates (inositol pyrophosphates, X-InsP7) are a family of second messengers with important roles in eukaryotic biology. Their chemical synthesis and modification remains a challenging task due to the high density of phosphate groups arranged around the myo-inositol core. Here, a novel approach is presented that facilitates the incorporation of the diphosphate in the 2-position (2-InsP7) and that enables the introduction of a photocage subunit.

Pharmacological tools-'chemical probes'-that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes 'high-energy' inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z' = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known-or conjectured based on their structures-to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1-10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.

Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals.

Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.

The propagation of information inside cells is achieved in nature by small diffusible molecules. Many of these secondary messengers are highly charged species derived from myo -inositol. The diphosphoinositol polyphosphates (X-PP- myo -InsP 5 ) represent a novel group of secondary messengers that regulate various important biological processes. Depending on the phosphorylation pattern, these molecules can occur in symmetric or nonsymmetric forms. In a recent study, we have shown the potential of C 2 symmetric phosphoramidites as reagents for desymmetrizing myo -inositol and for obtaining all four possible nonsymmetric X-PP- myo -InsP 5 stereoisomers in enantiomerically pure form. These results and their potential implications for the design of tools for chemical biology are discussed.

The ubiquitous mammalian signaling molecule bis-diphosphoinositol tetrakisphosphate (1,5-(PP)2 -myo-InsP4 , or InsP8 ) displays the most congested three-dimensional array of phosphate groups found in nature. The high charge density, the accumulation of unstable P-anhydrides and P-esters, the lack of UV absorbance, and low levels of optical rotation constitute severe obstacles to its synthesis, characterization, and purification. Herein, we describe the first procedure for the synthesis of enantiopure 1,5-(PP)2 -myo-InsP4 and 3,5-(PP)2 -myo-InsP4 utilizing a C2 -symmetric P-amidite for desymmetrization and concomitant phosphitylation followed by a one-pot bidirectional P-anhydride-forming reaction that combines sixteen chemical transformations with high efficiency. The configuration of these materials is unambiguously shown by subsequent X-ray analyses of both enantiomers after being individually soaked into crystals of the kinase domain of human diphosphoinositol pentakisphosphate kinase 2.

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised

Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] is “sensed” by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) as follows: during a period of Pi starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP7 levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219–231. Hitherto, such Pi sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP7 and ATP decrease upon [Pi] starvation and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP8 response, we characterized kinetic properties of the bifunctional 5-InsP7 kinase/InsP8 phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP8 in this case) to fluctuate independently of varying precursor (i.e. 5-InsP7) pool size. Moreover, we report that InsP8 phosphatase activities of PPIP5Ks are strongly inhibited by Pi (40–90% within the 0–1 mm range). For PPIP5K2, Pi sensing by InsP8 is amplified by a 2-fold activation of 5-InsP7 kinase activity by Pi within the 0–5 mm range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling, regulating InsP8 turnover independently of 5-InsP7, in response to fluctuations in extracellular supply of a key nutrient.

Inositol pyrophosphates (PP-InsPs) are “energetic” intracellular signals that are ubiquitous in animals, plants, and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution, reaction mechanisms, and regulation. Here, we describe the 2.35-Å-resolution structure of the catalytic core of Siw14, a 5-PP-InsP phosphatase from Saccharomyces cerevisiae and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses, thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs, but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. (a) The catalytic P-loop with the CX(5)R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the α5 and α6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the α5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. (c) The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis.

The 5-diphosphoinositol pentakisphosphate (5-InsP7) and bisdiphosphoinositol tetrakisphosphate (InsP8) are “energetic” inositol pyrophosphate signaling molecules that regulate bioenergetic homeostasis. Inositol pyrophosphate levels are regulated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks); these are large modular proteins that host a kinase domain (which phosphorylates 5-InsP7 to InsP8), a phosphatase domain that catalyzes the reverse reaction, and a polyphosphoinositide-binding domain (PBD). Here, we describe new interactions between these three domains in the context of full-length human PPIP5K1. We determine that InsP7 kinase activity is dominant when PPIP5K1 is expressed in intact cells; in contrast, we found that InsP8 phosphatase activity prevails when the enzyme is isolated from its cellular environment. We approach a reconciliation of this disparity by showing that cellular InsP8 phosphatase activity is inhibited by C8-PtdIns(4,5)P2 (IC50 ~40 μM). We recapitulate this phosphatase inhibition with natural PtdIns(4,5)P2 that was incorporated into large unilamellar vesicles. Additionally, PtdIns(4,5)P2 increases net InsP7 kinase activity 5-fold. We demonstrate that PtdIns(4,5)P2 is not itself a phosphatase substrate; its inhibition of InsP8 phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the PBD. Finally, we discuss the significance of PtdIns(4,5)P2 as a novel regulator of PPIP5K1, in relation to compartmentalization of InsP7/InsP8 signaling in vivo.