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• W2949492893 abstract "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. 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. Phosphate homeostasis is crucial for growth and survival, because virtually all biochemical processes utilize phosphate. In mammals, a complex hormonal regulatory network involving intestines, bones, and kidneys regulates serum phosphate concentration (1Michigami T. Kawai M. Yamazaki M. Ozono K. Phosphate as a signaling molecule and its sensing mechanism.Physiol. Rev. 2018; 98 (30109818): 2317-234810.1152/physrev.00022.2017Crossref PubMed Scopus (80) Google Scholar). Excessive serum phosphate (hyperphosphatemia) has been linked to cardiovascular disease and increased mortality in chronic kidney disease patients, whereas hypophosphatemia can result in a form of rickets. Serum phosphate levels are maintained by FGF23-αKlotho, parathyroid hormone, and vitamin D signaling. Many cell types have been shown to transcriptionally respond to changes in extracellular phosphate concentrations, and the phosphate uptake transporter Pit1/SLC20A1 has been proposed to sense extracellular phosphate. However, how phosphate homeostasis is maintained at the cellular level remains a poorly studied area. Analysis of yeast and plant genomes has revealed that the SPX protein domain (after SYG1, Pho81, and XPR1, Pfam: PF03105) is found in many proteins that regulate phosphate metabolism (2Azevedo C. Saiardi A. Eukaryotic phosphate homeostasis: the inositol pyrophosphate perspective.Trends Biochem. Sci. 2016; 42 (27876550): 219-231Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The Arabidopsis thaliana (thale cress) genome encodes 20 proteins containing SPX domains, whereas the Saccharomyces cerevisiae (budding yeast) genome contains 10 SPX proteins. Four of these, Vtc2–5, form part of the VTC complex that spans the vacuolar membrane. This complex synthesizes the linear polymer inorganic polyphosphate (polyP) 2The abbreviations used are: polyPpolymer inorganic polyphosphatePiinorganic phosphateAECadenylate energy chargeIP6inositol hexakisphosphate, phytic acidIP7diphosphoinositol pentakisphosphateIP8bisdiphosphoinositol tetrakisphosphatePP-IPsinositol pyrophosphatesPP-IP4diphosphoinositol tetrakisphosphateAMPKAMP-activated kinaseSRBsulforhodamine BDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumqPCRquantitative PCRFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazoneAntAantimycin ADKOdouble knockoutANOVAanalysis of variance. that functions as the main phosphate storage molecule in yeast (3Hothorn M. Neumann H. Lenherr E.D. Wehner M. Rybin V. Hassa P.O. Uttenweiler A. Reinhard M. Schmidt A. Seiler J. Ladurner A.G. Herrmann C. Scheffzek K. Mayer A. Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase.Science. 2009; 324: 513-51610.1126/science.1168120Crossref PubMed Scopus (211) Google Scholar). It is now known that VTC polyP synthesis requires binding of an inositol pyrophosphate, specifically the 5-diphosphoinositol pentakisphosphate (5PP-IP5; 5-IP7) isomer of IP7, to the VTC SPX domains (4Wild R. Gerasimaite R. Jung J.-Y. Truffault V. Pavlovic I. Schmidt A. Saiardi A. Jessen H.J. Poirier Y. Hothorn M. Mayer A. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.Science. 2016; 352 (27080106): 986-99010.1126/science.aad9858Crossref PubMed Scopus (315) Google Scholar, 5Gerasimaite R. Pavlovic I. Capolicchio S. Hofer A. Schmidt A. Jessen H.J. Mayer A. Inositol pyrophosphate specificity of the SPX-dependent polyphosphate polymerase VTC.ACS Chem. Biol. 2017; 12 (28186404): 648-65310.1021/acschembio.7b00026Crossref PubMed Scopus (55) Google Scholar), explaining the observation that yeast devoid of IP7 lack polyP (6Lonetti A. Szijgyarto Z. Bosch D. Loss O. Azevedo C. Saiardi A. Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases.J. Biol. Chem. 2011; 286: 31966-3197410.1074/jbc.M111.266320Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). IP7-SPX binding also promotes the interaction of two rice phosphate-regulated transcription factors, OsSPX4 (an SPX domain-containing protein) and OsPHR2. Electrophysiological measurements of the parasite Trypanosoma brucei SPX protein TbPho91, and its yeast homolog Pho91, suggest IP7 as a regulator of their phosphate transport activities (7Potapenko E. Cordeiro C.D. Huang G. Storey M. Wittwer C. Dutta A.K. Jessen H.J. Starai V.J. Docampo R. 5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles.J. Biol. Chem. 2018; 293: 19101-1911210.1074/jbc.RA118.005884Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). polymer inorganic polyphosphate inorganic phosphate adenylate energy charge inositol hexakisphosphate, phytic acid diphosphoinositol pentakisphosphate bisdiphosphoinositol tetrakisphosphate inositol pyrophosphates diphosphoinositol tetrakisphosphate AMP-activated kinase sulforhodamine B Dulbecco's modified Eagle's medium fetal bovine serum quantitative PCR carbonyl cyanide p-trifluoromethoxyphenylhydrazone antimycin A double knockout analysis of variance. Inositol pyrophosphates (PP-IPs) are myo-inositol–derived signaling molecules ubiquitous in eukaryotic cells (8Wilson M.S. Livermore T.M. Saiardi A. Inositol pyrophosphates: between signalling and metabolism.Biochem. J. 2013; 379 (23725456): 369-379Crossref Scopus (183) Google Scholar). They are defined and distinguished from other inositol phosphates by the presence of at least one phosphoanhydride bond. The best characterized, 5-IP7, whose phosphoanhydride bond is at the 5-position of the myo-inositol carbon ring (8Wilson M.S. Livermore T.M. Saiardi A. Inositol pyrophosphates: between signalling and metabolism.Biochem. J. 2013; 379 (23725456): 369-379Crossref Scopus (183) Google Scholar), is synthesized by IP6K enzymes from inositol hexakisphosphate (IP6; phytic acid). The IP6Ks are catalytically flexible: they can use IP5 as substrate to generate 5PP-IP4, whereas in the presence of ADP they can dephosphorylate IP6 to I(2,3,4,5,6)P5 (9Wundenberg T. Grabinski N. Lin H. Mayr G.W. Discovery of InsP6-kinases as InsP6-dephosphorylating enzymes provides a new mechanism of cytosolic InsP6 degradation driven by the cellular ATP/ADP ratio.Biochem. J. 2014; 462 (24865181): 173-18410.1042/BJ20130992Crossref PubMed Scopus (40) Google Scholar). Eukaryote genomes also contain another class of kinase, namely the PPIP5Ks, able to synthesize PP-IPs. These enzymes preferentially act on 5-IP7, converting it to 1,5(PP)2-IP4 (called IP8) (10Fridy P.C. Otto J.C. Dollins D.E. York J.D. Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.J, Biol. Chem. 2007; 282: 30754-3076210.1074/jbc.M704656200Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 11Choi J.H. Williams J. Cho J. Falck J.R. Shears S.B. Purification sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress.J. Biol. Chem. 2007; 282: 30763-3077510.1074/jbc.M704655200Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Budding yeast has one IP6K enzyme named Kcs1, whereas mammalian genomes encode three IP6K isoforms: IP6K1, IP6K2, and IP6K3. Studies using kcs1Δ yeast or individual IP6Ks KO mice have linked PP-IPs to a plethora of cellular activities (12Burton A. Hu X. Saiardi A. Are inositol pyrophosphates signalling molecules?.J. Cell Physiol. 2009; 220: 8-1510.1002/jcp.21763Crossref PubMed Scopus (71) Google Scholar), leading to the suggestion that PP-IPs act as “metabolic messenger” (13Shears S.B. Diphosphoinositol polyphosphates: metabolic messengers?.Mol. Pharmacol. 2009; 76 (19439500): 236-25210.1124/mol.109.055897Crossref PubMed Scopus (122) Google Scholar): a signaling molecule involved in the regulation of metabolic homeostasis (14Wundenberg T. Mayr G.W. Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis.Biol. Chem. 2012; 393 (22944697): 979-998Crossref PubMed Scopus (64) Google Scholar). Besides reduced polyP levels (6Lonetti A. Szijgyarto Z. Bosch D. Loss O. Azevedo C. Saiardi A. Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases.J. Biol. Chem. 2011; 286: 31966-3197410.1074/jbc.M111.266320Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), kcs1Δ yeast show reduced uptake of phosphate from the culture medium (15Saiardi A. Bhandari R. Resnick A.C. Snowman A.M. Snyder S.H. Phosphorylation of proteins by inositol pyrophosphates.Science. 2004; 306 (15604408): 2101-210510.1126/science.1103344Crossref PubMed Scopus (241) Google Scholar). This appears to be an evolutionarily conserved phenotype, because mammalian IP6K2 was initially discovered as a protein that could stimulate phosphate (Pi) uptake into Xenopus oocytes, and was called PiUS (Pi Uptake Stimulator) before its enzymatic abilities were discovered (16Schell M.J. Letcher A.J. Brearley C.A. Biber J. Murer H. Irvine R.F. PiUS (Pi uptake stimulator) is an inositol hexakisphosphate kinase.FEBS Lett. 1999; 461: 169-17210.1016/S0014-5793(99)01462-3Crossref PubMed Scopus (74) Google Scholar, 17Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.Curr. Biol. 1999; 9 (10574768): 1323-132610.1016/S0960-9822(00)80055-XAbstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar18Norbis F. Boll M. Stange G. Markovich D. Verrey F. Biber J. Murer H. Identification of a cDNA/protein leading to an increased Pi-uptake in Xenopus laevis oocytes.J. Membr. Biol. 1997; 156: 19-2410.1007/s002329900183Crossref PubMed Scopus (45) Google Scholar). Furthermore, two single-nucleotide polymorphisms within the human IP6K3 gene locus are associated with differences in serum phosphate concentration (19Kestenbaum B. et al.Common genetic variants associate with serum phosphorus concentration.J. Am. Soc. Nephrol. 2010; 21: 1223-123210.1681/ASN.2009111104Crossref PubMed Scopus (105) Google Scholar). Unlike yeast or plants, mammalian genomes contain a single SPX domain-containing protein. Localized at the plasma membrane, XPR1 was originally characterized as a retroviral receptor (Xenotropic and Polytropic retrovirus Receptor 1), but is functionally a phosphate exporter (20Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the current study we aimed to investigate if and how PP-IPs regulate intracellular phosphate homeostasis in mammalian cells. Most previous work has used cells knocked out for only one IP6K at a time, generating cells with a reduction rather than complete depletion of PP-IPs levels. IP6K1 and IP6K2 have a wide and overlapping tissue distribution, whereas IP6K3 is highly expressed in skeletal muscle (21Moritoh Y. Oka M. Yasuhara Y. Hozumi H. Iwachidow K. Fuse H. Tozawa R. Inositol hexakisphosphate kinase 3 regulates metabolism and lifespan in mice.Sci. Rep. 2016; 6 (27577108)3207210.1038/srep32072Crossref PubMed Scopus (45) Google Scholar). We used CRISPR and the human colon carcinoma line HCT116 to create a cell line truly devoid of PP-IPs by disrupting both IP6K1 and IP6K2. These DKO cells showed an increased amount of ATP as well as increased intracellular free phosphate. Conversely, release as well as uptake of radioactive phosphate was reduced. Knockdown of XPR1 inhibited [32Pi] release in WT cells, but had no effect in DKO cells, demonstrating that PP-IPs regulate phosphate export through XPR1. To study the role that PP-IPs play in mammalian phosphate homeostasis, we generated cells devoid of this small molecule messenger. Mammalian genomes possess three IP6K homologs. The IP6K1 and IP6K2 genes are located close together on the same chromosome (located at chromosome 3p21.31 in humans; in mice, chromosome 9;9 F1 and 9;9 F2 for ip6k1 and ip6k2, respectively) (Fig. 1A). Therefore, crossing the ip6k1−/− and ip6k2−/− mice is unlikely to generate an ip6k1−/−ip6k2−/− DKO mouse because of linkage. Instead, we used CRISPR to create a cell line without PP-IPs. IP6K3 is expressed mainly in muscle cells (21Moritoh Y. Oka M. Yasuhara Y. Hozumi H. Iwachidow K. Fuse H. Tozawa R. Inositol hexakisphosphate kinase 3 regulates metabolism and lifespan in mice.Sci. Rep. 2016; 6 (27577108)3207210.1038/srep32072Crossref PubMed Scopus (45) Google Scholar), and indeed the cell lines we tested, which were not muscle-derived, contained no or a very low amount of IP6K3 mRNA, whereas IP6K1 and IP6K2 were widely co-expressed (Fig. S1A). We chose to use the human colon carcinoma cell line HCT116 as it is near-diploid (22Knutsen T. Padilla-Nash H.M. Wangsa D. Barenboim-Stapleton L. Camps J. McNeil N. Difilippantonio M.J. Ried T. Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.Genes Chromosomes Cancer. 2010; 49 (19927377): 204-223PubMed Google Scholar), expresses IP6K1 and IP6K2 only, and has easily detectable levels of IP7 and IP8 (23Wilson M.S. Bulley S.J. Pisani F. Irvine R.F. Saiardi A. A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine.Open Biol. 2015; 5 (25808508)15001410.1098/rsob.150014Crossref PubMed Scopus (89) Google Scholar). We targeted IP6K1 and IP6K2 using guide RNAs against exon 5 of both genes to disrupt the inositol-binding motifs (Fig. 1B) (17Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.Curr. Biol. 1999; 9 (10574768): 1323-132610.1016/S0960-9822(00)80055-XAbstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). No IP6K1 or IP6K2 protein was detectable in DKO IP6K1−/−IP6K2−/− lines (Fig. 1C), which also contained significantly less IP6K1 and IP6K2 mRNA than WT cells (Fig. 1D). We attempted to analyze IP6K3 mRNA, in case this homolog was up-regulated to compensate, but were unable to detect any in the knockout or WT cells. We then analyzed the effect of loss of IP6Ks on the cellular inositol phosphate profile. Similar results were obtained with both TiO2 beads purification PAGE analysis (Fig. 1E) and [3H]inositol labeling SAX-HPLC (Fig. 1F) techniques. Single IP6K knockout lines still showed detectable levels of IP7. In IP6K2−/− cells, IP7 was significantly reduced compared with WT but was still present, whereas the IP6K1−/− line did not have significantly less IP7 than WT. No IP7 or IP8 was detectable in the DKO cells, demonstrating that, while IP6K2 appears to contribute most IP7 in HCT116 cells, knockout of both IP6K1 and IP6K2 is necessary to completely deplete these PP-IPs. We consequently focused our attention on the DKO cell lines. These cells showed a normal level of IP6 and the major IP5 isoform I(1,3,4,5,6)P5 (Fig. 2). Two smaller peaks were seen eluting between I(1,3,4,5,6)P5 and IP6. One was identified by coelution with standard as the alternative IP6K product I(2,3,4,5,6)P5 or its indistinguishable stereoisomer I(1,2,4,5,6)P5 (9Wundenberg T. Grabinski N. Lin H. Mayr G.W. Discovery of InsP6-kinases as InsP6-dephosphorylating enzymes provides a new mechanism of cytosolic InsP6 degradation driven by the cellular ATP/ADP ratio.Biochem. J. 2014; 462 (24865181): 173-18410.1042/BJ20130992Crossref PubMed Scopus (40) Google Scholar). As this peak increased in the DKO cells (Fig. 2B), in vivo one or both of these isomers must be synthesized through an IP6K-independent but regulated route. The second peak, strongly reduced in the DKO cells, coeluted with a standard of the IP6K product 5PP-IP4. The remaining signal suggests coelution of an unknown species, probably another IP5 isomer, that we have named IPx. Morphologically, the DKO cells appeared similar to the WT cells (Fig. 1G), and there was no significant difference in cell growth (Fig. 1H). Treatment of mammalian cells with the phosphatase inhibitor sodium fluoride is known to increase levels of PP-IPs by inhibiting their turnover (24Glennon M.C. Shears S.B. Turnover of inositol pentakisphosphates, inositol hexakisphosphate and diphosphoinositol polyphosphates in primary cultured hepatocytes.Biochem. J. 1993; 293 (8343137): 583-59010.1042/bj2930583Crossref PubMed Scopus (97) Google Scholar). Fluoride treatment of WT cells resulted in 5-IP7 and IP8 accumulation as expected (Fig. S1B). A band migrating slightly faster than IP6 is also seen, likely 5PP-IP4. However, the DKO cells accumulated solely the PPIP5K product 1-IP7, which migrates more slowly. In WT cells 1-IP7 represents <2% of total IP7 (25Gu C. Wilson M.S. Jessen H.J. Saiardi A. Shears S.B. Inositol pyrophosphate profiling of two HCT116 cell lines uncovers variation in InsP8 levels.PLoS ONE. 2016; 11 (27788189)e016528610.1371/journal.pone.0165286Crossref PubMed Scopus (27) Google Scholar). Expression of the mammalian PPIP5K isoforms PPIP5K1 and PPIP5K2 was unchanged in IP6K KO cells (Fig. S1C). Previous work found that kcs1Δ yeast and IP6K1−/− murine embryonic fibroblasts have defects in mitochondrial function (26Szijgyarto Z. Garedew A. Azevedo C. Saiardi A. Influence of inositol pyrophosphates on cellular energy dynamics.Science. 2011; 334 (22076377): 802-80510.1126/science.1211908Crossref PubMed Scopus (162) Google Scholar). Surprisingly, we did not find any difference between WT and DKO cells using respirometry (Fig. 3A). The mitochondria looked similar following MitoTracker Deep Red staining (Fig. 3B), and expression of various electron transport complex subunits was unchanged (Fig. 3C). It is possible that the different result seen in murine embryonic fibroblasts demonstrates cell type specificity for IP7 function. As discussed above, PP-IPs regulate phosphate storage and buffering in budding yeast by controlling polyP production. The presence, abundance, and nature of polyP in mammalian cells remains an open question, and likely will be until the relevant mammalian polyP enzymology is discovered. No true polyP null or overexpression cells are available to act as controls. We attempted to detect polyP in WT and DKO cells. Phenol extraction followed by PAGE and 4′,6-diamidino-2-phenylindole (DAPI) staining did not reveal any polyP characteristic ladders or smears (Fig. S2A). We then tried radioactive [32Pi] labeling to maximize sensitivity. Cells were starved of phosphate for 24 h, on the assumption that if polyP was present it would be degraded during this period to provide phosphate. Phosphate was then restored, along with [32Pi], for a further 24 h, which would stimulate synthesis of radiolabeled [32Pi]polyP. Visualization of PAGE-resolved extracts by toluidine blue and autoradiography also failed to show any polyP-like signal (Fig. S2B). HCT116 cells may therefore not possess polyP; if it exists there, it is both low abundance and very short chain, requiring novel extraction procedures and detection methods more sensitive than radioactive labeling. The absence of polyP led us to theorize that other phosphate-rich and abundant molecules, such as ATP, might work as phosphate buffers in mammalian cells. Intracellular ATP exists at millimolar concentrations (27Yoshida T. Kakizuka A. Imamura H. BTeam a novel BRET-based biosensor for the accurate quantification of ATP concentration within living cells.Sci. Rep. 2016; 6 (28000761)3961810.1038/srep39618Crossref PubMed Scopus (62) Google Scholar), representing a large portion of a cell’s phosphate content. Using ion-pairing HPLC we simultaneously and quantitatively measured ATP, ADP, and AMP in cell extracts (Fig. 3D). A 1.7-fold increase in amount of ATP was seen for DKO cells (Table S1), but there were no significant alterations in AMP or ADP level. The relative proportions of AMP, ADP, and ATP can be combined into one value by calculating the adenylate energy charge (AEC; (ATP + 0.5 ADP)/(ATP + ADP + AMP)). The DKO cells had significantly higher AEC ratio (Fig. 3E, Table S1). No significant alterations in ATP level or AEC were found for single IP6K1−/− and IP6K2−/− cell lines. Cellular sensing of energy, via sensing of AMP:ATP and ADP:ATP ratios, is performed by the AMPK (AMP-activated kinase) complex. Phosphorylation of AMPK’s α subunit at Thr172 is required for full activity, and this is promoted by AMP binding (28Garcia D. Shaw R.J. AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance.Mol. Cell. 2017; 66 (28622524): 789-80010.1016/j.molcel.2017.05.032Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). The increase in AEC in DKO cells, meaning proportionately less AMP, was reflected in a >0.6-fold reduction in AMPK phosphorylation (Fig. 3F). To confirm the ATP increase following PP-IPs cellular depletion in another cell type and by using a different experimental approach, we used the yeast 5-pyrophosphatase Siw14 (29Steidle E.A. Chong L.S. Wu M. Crooke E. Fiedler D. Resnick A.C. Rolfes R.J. A novel inositol pyrophosphate phosphatase in Saccharomyces cerevisiae: Siw14 selectively cleaves the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5).J. Biol. Chem. 2016; 291: 6772-678310.1074/jbc.M116.714907Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This enzyme has no mammalian homolog. We transfected HeLa cells with a plasmid encoding either the WT humanized Myc-Siw14 or pyrophosphatase-dead C214S mutant. Overexpression of the active, but not the dead, Myc-Siw14 depleted IP7 and IP8 levels (Fig. S3A). As with the HCT116 DKO stable cell line, which experiences a chronic loss of PP-IPs, acute depletion by transient transfection in HeLa cells also caused a significant increase in ATP cellular level (Fig. S3B). In this case, levels of ADP and AMP were also higher when PP-IPs were depleted. The increased adenylate pools in DKO likely reflects an increase in total cellular phosphate content. We next investigated if free phosphate levels were also affected. Indeed, DKO cells contained 1.3-fold more free phosphate compared with WT cells (WT 10.0 ± 0.9 versus DKO 13.1 ± 1.7 pmol of phosphate/μg of protein; Fig. 4A). Starving the cells of phosphate for 24 h decreased free intracellular phosphate in both lines to 0.4-fold of the phosphate-replete value; there was no significant difference in phosphate concentration in starved cells. The absence of PP-IPs in DKO cells therefore results in an increase in both free phosphate and bound phosphate, in the form of adenylates. Such phosphate overload could negatively influence phosphate flux in and out of the cells. To test this hypothesis, the steady-state results were complemented by [32Pi] pulse labeling experiments to analyze flux (Fig. 4, B and C). To maximize physiological relevance, culture medium containing the usual phosphate concentration was used, rather than phosphate-free medium. Both [32Pi] uptake and release were significantly reduced in the DKO cells: 0.7-fold [32Pi] was taken up, and 0.6-fold [32Pi] released back into the medium, validating our hypothesis. Release of [32Pi] from labeled cells into the medium is XPR1-dependent (20Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This protein is the only identified mammalian phosphate exporter, but several phosphate importers (sodium-phosphate cotransporters) are known, across three classes: SLC17, SLC34, and SLC20 (1Michigami T. Kawai M. Yamazaki M. Ozono K. Phosphate as a signaling molecule and its sensing mechanism.Physiol. Rev. 2018; 98 (30109818): 2317-234810.1152/physrev.00022.2017Crossref PubMed Scopus (80) Google Scholar). We were only able to detect mRNA for SLC20A1/Pit1 in HCT116 cells. There was no statistically significant difference in expression of Pit1 or XPR1 (Fig. S4, A and B) between WT and DKO cells. XPR1 is also the only mammalian SPX domain-containing protein. As PP-IPs are known to regulate the activity of SPX proteins from other systems, we were interested in a closer look at the reduced phosphate efflux phenotype. We knocked down XPR1 using siRNA. Commercial antibodies for XPR1 are unable to detect endogenous protein by Western blotting. Therefore, we relied on RT-qPCR to record a reduction in XPR1 mRNA (Fig. 5A). Transient knockdown of XPR1 significantly reduced [32Pi] release 0.8-fold in WT HCT116 cells (Fig. 5B), again confirming its role as phosphate exporter (20Giovannini D. Touhami J. Charnet P. Sitbon M. Battini J.L. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.Cell Rep. 2013; 3 (23791524): 1866-187310.1016/j.celrep.2013.05.035Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, siXPR1 did not further affect the low [32Pi] release from DKO cells. This shows that in WT cells phosphate is exported through XPR1 in an IP6K1/2-generated PP-IPs-dependent manner. The ability of XPR1’s SPX domain to bind IP6 has been shown by NMR (4Wild R. Gerasimaite R. Jung J.-Y. Truffault V. Pavlovic I. Schmidt A. Saiardi A. Jessen H.J. Poirier Y. Hothorn M. Mayer A. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains.Science. 2016; 352 (27080106): 986-99010.1126/science.aad9858Crossref PubMed Scopus (315) Google Scholar). To investigate if PP-IPs such as IP7 are XPR1 ligands, we incubated purified SPXXPR1 with [3H]IP6 for a competition-binding assay (Fig. 5C). Titration of unlabeled IP7 reduced the radioactive counts, meaning that it was able to compete with the radioactive [3H]IP6 for binding. We observed similar IC50 values for competing for binding for IP6, 1-IP7, and 5-IP7 of ∼9.8, 8.4, and 4.2 μm, respectively, whereas I(1,3,4,5,6)P5 displaced IP6 with a lower affinity. Therefore, IP7 can bind directly to this SPX protein. Similar affinities for IP6 and IP7 appears to be a common characteristic of SPX domains from various proteins. However, as seen here for XPR1, studies of the yeast VTC complex also revealed functional selectivity for IP7 over IP6 (5Gerasimaite R. Pavlovic I. Capolicchio S. Hofer A. Schmidt A. Jessen H.J. Mayer A. Inositol pyrophosphate specificity of the SPX-dependent polyphosphate polymerase VTC.ACS Chem. Biol. 2017; 12 (28186404): 648-65310.1021/acschembio.7b00026Crossref PubMed Scopus (55) Google Scholar). To uncover a role for PP-IPs in mammalian phosp" @default.
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• W2949492893 cites W1981979006 @default.
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• W2949492893 cites W2162217347 @default.
• W2949492893 cites W2164269498 @default.
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