Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels.
ABSTRACT: 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 <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function.
Project description: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 ?M, 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 ?M) and UNC10225498 (Kd = 1.37 ± 0.03 ?M). These Kd values lie within the 1-10 ?M 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 ?M, 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.
Project description:The inositol pyrophosphates 5-InsP<sub>7</sub> (diphosphoinositol pentakisphosphate) and 1,5-InsP<sub>8</sub> (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-InsP<sub>8</sub> and raises 5-InsP<sub>7</sub> 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-InsP<sub>7</sub>] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP<sub>7</sub>] provides direct support for an energy-sensing attribute (i.e., 1 mM <i>K</i><sub>m</sub> for ATP) of the 5-InsP<sub>7</sub>-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP<sub>7</sub> levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in <i>PPIP5K</i><sup>-/-</sup> HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP<sub>8</sub> and 5-InsP<sub>7</sub> as drivers of the hypermetabolic and p53-elevated phenotypes, we used <i>IP6K2</i> RNAi and the pan-IP6K inhibitor, <i>N</i>2-(<i>m</i>-trifluorobenzyl), <i>N</i>6-(<i>p</i>-nitrobenzyl) purine (TNP), to return 5-InsP<sub>7</sub> levels in <i>PPIP5K</i><sup>-/-</sup> cells to those of WT cells without rescuing 1,5-InsP<sub>8</sub> levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP<sub>7</sub> regulates p53 expression, whereas 1,5-InsP<sub>8</sub> regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP<sub>8</sub> to bioenergetic homeostasis.
Project description: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.
Project description: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. 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. 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.
Project description: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, <i>inter alia</i>, 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-InsP<sub>5</sub>, the major cellular inositol pyrophosphate, oscillations of intracellular Ca<sup>2+</sup> levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP<sub>5</sub>, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca<sup>2+</sup>]<sub>i</sub> oscillations, a key element in triggering exocytosis and secretion in β-cells.
Project description: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 (TiO2) 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 InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.
Project description:Inositol pyrophosphates (PP-InsPs), including diphospho-<i>myo</i>-inositol pentakisphosphate (5-InsP<sub>7</sub>) and bis-diphospho-<i>myo</i>-inositol tetrakisphosphate (1,5-InsP<sub>8</sub>), 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-InsP<sub>7</sub> analogue (<i>i.e.</i>, 5-FAM-InsP<sub>7</sub>) 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 10<sup>6</sup> cultured cells. Finally, to interrogate the biological value of these procedures, we delivered 1,5-InsP<sub>8</sub> into HCT116 cells and showed it to dose-dependently stimulate the rate of [<sup>33</sup>P]-Pi uptake; these observations reveal a rheostatic range of concentrations over which 1,5-InsP<sub>8</sub> is biologically functional in Pi homeostasis.
Project description:Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.
Project description: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-InsP<sub>7</sub> (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP<sub>7</sub> 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 <i>PPIP5Ks</i> (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., <i>PPIP5K</i> KO), which elevates cellular 5-InsP<sub>7</sub> levels by two- to threefold (i.e., within the physiological rheostatic range). The <i>PPIP5K</i> KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP<sub>7</sub> 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-InsP<sub>7</sub> analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP<sub>7</sub> 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.
Project description:Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are an important group of metabolites and mediate a wide range of processes in eukaryotic cells. To elucidate the functions of these molecules, robust techniques for the characterization of inositol phosphate metabolism are required, both at the biochemical and the cellular level. Here, a new tool-set is reported, which employs uniformly <sup>13</sup>C-labeled compounds ([<sup>13</sup>C<sub>6</sub>]<i>myo</i>-inositol, [<sup>13</sup>C<sub>6</sub>]InsP<sub>5</sub>, [<sup>13</sup>C<sub>6</sub>]InsP<sub>6</sub>, and [<sup>13</sup>C<sub>6</sub>]5PP-InsP<sub>5</sub>), in combination with commonly accessible NMR technology. This approach permitted the detection and quantification of InsPs and PP-InsPs within complex mixtures and at physiological concentrations. Specifically, the enzymatic activity of IP6K1 could be monitored <i>in vitro</i> in real time. Metabolic labeling of mammalian cells with [<sup>13</sup>C<sub>6</sub>]<i>myo</i>-inositol enabled the analysis of cellular pools of InsPs and PP-InsPs, and uncovered high concentrations of 5PP-InsP<sub>5</sub> in HCT116 cells, especially in response to genetic and pharmacological perturbation. The reported method greatly facilitates the analysis of this otherwise spectroscopically silent group of molecules, and holds great promise to comprehensively analyze inositol-based signaling molecules under normal and pathological conditions.