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

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

Co-culturing experiments reveal the uptake of myo-inositol phosphate synthase (EC 5.5.1.4) in an inositol auxotroph of Saccharomyces cerevisiae.

BACKGROUND: Myo-Inositol Phosphate Synthase (MIP) catalyzes the conversion of glucose 6- phosphate into inositol phosphate, an essential nutrient and cell signaling molecule. Data obtained, first in bovine brain and later in plants, established MIP expression in organelles and in extracellular environments. A physiological role for secreted MIP has remained elusive since its first detection in intercellular space. To provide further insight into the role of MIP in intercellular milieus, we tested the hypothesis that MIP may function as a growth factor, synthesizing inositol phosphate in intercellular locations requiring, but lacking ability to produce or transport adequate quantities of the cell-cell communicator. This idea was experimentally challenged, utilizing a Saccharomyces cerevisiae inositol auxotroph with no MIP enzyme, permeable membranes with a 0.4 µm pore size, and cellular supernatants as external sources of inositol isolated from S. cerevisiae cells containing either wild-type enzyme (Wt-MIP), no MIP enzyme, auxotroph (Aux), or a green fluorescent protein (GFP) tagged reporter enzyme (MIP- GFP) in co- culturing experiments. RESULTS: Resulting cell densities and microscopic studies with corroborating biochemical and molecular analyses, documented sustained growth of Aux cells in cellular supernatant, concomitant with the uptakeof MIP, detected as MIP-GFP reporter enzyme. These findings revealed previously unknown functions, suggesting that the enzyme can: (1) move into and out of intercellular space, (2) traverse cell walls, and (3) act as a growth factor to promote cellular proliferation of an inositol requiring cell. CONCLUSIONS: Co-culturing experiments, designed to test a probable function for MIP secreted in extracellular vesicles, uncovered previously unknown functions for the enzyme and advanced current knowledge concerning spatial control of inositol phosphate biosynthesis. Most importantly, resulting data identified an extracellular vesicle (a non-viral vector) that is capable of synthesizing and transporting inositol phosphate, a biological activity that can be used to enhance specificity of current inositol phosphate therapeutics.

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

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

Bile acids inhibit cholinergic constriction in proximal and peripheral airways from humans and rodents.

Duodenogastroesophageal reflux (DGER) is associated with chronic lung disease. Bile acids (BAs) are established markers of DGER aspiration and are important risk factors for reduced post-transplant lung allograft survival by disrupting the organ-specific innate immunity, facilitating airway infection and allograft failure. However, it is unknown whether BAs also affect airway reactivity. We investigated the acute effects of 13 BAs detected in post-lung-transplant surveillance bronchial washings (BW) on airway contraction. We exposed precision-cut slices from human and mouse lungs to BAs and monitored dynamic changes in the cross-sectional luminal area of peripheral airways using video phase-contrast microscopy. We also used guinea pig tracheal rings in organ baths to study BA effects in proximal airway contraction induced by electrical field stimulation. We found that most secondary BAs at low micromolar concentrations strongly and reversibly relaxed smooth muscle and inhibited peripheral airway constriction induced by acetylcholine but not by noncholinergic bronchoconstrictors. Similarly, secondary BAs strongly inhibited cholinergic constrictions in tracheal rings. In contrast, TC-G 1005, a specific agonist of the BA receptor Takeda G protein-coupled receptor 5 (TGR5), did not cause airway relaxation, and Tgr5 deletion in knockout mice did not affect BA-induced relaxation, suggesting that this receptor is not involved. BAs inhibited acetylcholine-induced inositol phosphate synthesis in human airway smooth muscle cells overexpressing the muscarinic M3 receptor. Our results demonstrate that select BAs found in BW of patients with lung transplantation can affect airway reactivity by inhibiting the cholinergic contractile responses of the proximal and peripheral airways, possibly by acting as antagonists of M3 muscarinic receptors.

Agonism of the TMEM16A calcium-activated chloride channel modulates airway smooth muscle tone.

TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.

The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery.

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1.

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell-cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina.

mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5(-/-) mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5ß recruitment at the cytocortex.

BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5ß to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5ß or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5ß signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

Role of SAP97 in the regulation of 5-HT2AR endocytosis and signaling.

Serotonin (5-HT) interacts with a wide variety of 5-HT receptors (5-HTR) of which 5-HT2AR plays an important target for antidepressant and atypical antipsychotic drugs. The carboxyl-terminal tail of 5-HT2AR encodes a motif that mediates interactions with PSD-95/disc large/zona occludens (PDZ) domain-containing proteins. In the present study, we found that 5-HT2AR interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by coimmunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates. We found that 5-HT2AR expression results in the recruitment of SAP97 from the cytosol to the plasma membrane and that this recruitment is dependent on an intact 5-HT2AR PDZ binding motif. We also show that 5-HT2AR interacts with SAP97 using bioluminescence energy transfer and that overexpression of SAP97 retards 5-HT2AR endocytosis, while single hairpin RNA knockdown facilitates 5-HT2AR internalization. The knockdown of SAP97 in HEK 293 cells results in a reduction in the maximum efficacy for 5-HT2AR-stimulated inositol phosphate formation and that the deletion of the 5-HT2AR PDZ motif also impairs 5-HT2AR signaling. Similarly to what has been observed for the corticotropin-releasing factor receptor 1 (CRFR1), SAP97 expression is essential for 5-HT2AR-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation by a PDZ interaction-independent mechanism. Moreover, we find that SAP97 is not responsible for CRFR1-mediated sensitization of 5-HT2AR signaling. Taken together, our studies show that SAP97 plays a conserved role in regulating 5-HT2AR endocytosis and ERK1/2 signaling, but plays a novel role in regulating 5-HT2AR G protein coupling.

Inositol pyrophosphate synthesis by inositol hexakisphosphate kinase 1 is required for homologous recombination repair.

Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP(7)), are water-soluble inositol phosphates that contain high energy diphosphate moieties on the inositol ring. Inositol hexakisphosphate kinase 1 (IP6K1) participates in inositol pyrophosphate synthesis, converting inositol hexakisphosphate (IP(6)) to IP(7). In the present study, we show that mouse embryonic fibroblasts (MEFs) lacking IP6K1 exhibit impaired DNA damage repair via homologous recombination (HR). IP6K1 knock-out MEFs show decreased viability and reduced recovery after induction of DNA damage by the replication stress inducer, hydroxyurea, or the radiomimetic antibiotic, neocarzinostatin. Cells lacking IP6K1 arrest after genotoxic stress, and markers associated with DNA repair are recruited to DNA damage sites, indicating that HR repair is initiated in these cells. However, repair does not proceed to completion because these markers persist as nuclear foci long after drug removal. A fraction of IP6K1-deficient MEFs continues to proliferate despite the persistence of DNA damage, rendering the cells more susceptible to chromosomal aberrations. Expression of catalytically active but not inactive IP6K1 can restore the repair process in knock-out MEFs, implying that inositol pyrophosphates are required for HR-mediated repair. Our study therefore highlights inositol pyrophosphates as novel small molecule regulators of HR signaling in mammals.

Regulation of inositol metabolism is fine-tuned by inositol pyrophosphates in Saccharomyces cerevisiae.

Although inositol pyrophosphates have diverse roles in phosphate signaling and other important cellular processes, little is known about their functions in the biosynthesis of inositol and phospholipids. Here, we show that KCS1, which encodes an inositol pyrophosphate kinase, is a regulator of inositol metabolism. Deletion of KCS1, which blocks synthesis of inositol pyrophosphates on the 5-hydroxyl of the inositol ring, causes inositol auxotrophy and decreased intracellular inositol and phosphatidylinositol. These defects are caused by a profound decrease in transcription of INO1, which encodes myo-inositol-3-phosphate synthase. Expression of genes that function in glycolysis, transcription, and protein processing is not affected in kcs1Δ. Deletion of OPI1, the INO1 transcription repressor, does not fully rescue INO1 expression in kcs1Δ. Both the inositol pyrophosphate kinase and the basic leucine zipper domains of KCS1 are required for INO1 expression. Kcs1 is regulated in response to inositol, as Kcs1 protein levels are increased in response to inositol depletion. The Kcs1-catalyzed production of inositol pyrophosphates from inositol pentakisphosphate but not inositol hexakisphosphate is indispensable for optimal INO1 transcription. We conclude that INO1 transcription is fine-tuned by the synthesis of inositol pyrophosphates, and we propose a model in which modulation of Kcs1 controls INO1 transcription by regulating synthesis of inositol pyrophosphates.

Mannosylglycerate and di-myo-inositol phosphate have interchangeable roles during adaptation of Pyrococcus furiosus to heat stress.

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

Arginine transcriptional response does not require inositol phosphate synthesis.

Inositol phosphates are key signaling molecules affecting a large variety of cellular processes. Inositol-polyphosphate multikinase (IPMK) is a central component of the inositol phosphate biosynthetic routes, playing essential roles during development. IPMK phosphorylates inositol 1,4,5-trisphosphate to inositol tetrakisphosphate and subsequently to inositol pentakisphosphate and has also been described to function as a lipid kinase. Recently, a catalytically inactive mammalian IPMK was reported to be involved in nutrient signaling by way of mammalian target of rapamycin and AMP-activated protein kinase. In yeast, the IPMK homologue, Arg82, is the sole inositol-trisphosphate kinase. Arg82 has been extensively studied as part of the transcriptional complex regulating nitrogen sensing, in particular arginine metabolism. Whether this role requires Arg82 catalytic activity has long been a matter of contention. In this study, we developed a novel method for the real time study of promoter strength in vivo and used it to demonstrate that catalytically inactive Arg82 fully restored the arginine-dependent transcriptional response. We also showed that expression in yeast of catalytically active, but structurally very different, mammalian or plant IPMK homologue failed to restore arginine regulation. Our work indicates that inositol phosphates do not regulate arginine-dependent gene expression.

Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels.

G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gα(q/11). To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t(1/2)=77 ± 8.1 min) compared with TRH-R1 (t(1/2)=82 ± 12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gα(q/11) knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gα(q/11) induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.

Synthesis of inositol phosphate ligands of plant hormone-receptor complexes: pathways of inositol hexakisphosphate turnover.

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone-receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

Inositol pyrophosphate pyrotechnics.

Physiologic roles of highly phosphorylated inositol phosphates, including those containing pyrophosphate groups, have been the focus of much recent interest. In the April 6, 2007 issue of Science, two papers (Lee et al., 2007; Mulugu et al., 2007) demonstrate the occurrence of a novel inositol pyrophosphate molecule in yeast and elucidate its role in phosphate homeostasis.

Evolution of the biosynthesis of di-myo-inositol phosphate, a marker of adaptation to hot marine environments.

The synthesis of di-myo-inositol phosphate (DIP), a common compatible solute in hyperthermophiles, involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS). In most cases, both activities are present in a single gene product, but separate genes are also found in a few organisms. Genes for IPCT and DIPPS were found in the genomes of 33 organisms, all with thermophilic/hyperthermophilic lifestyles. Phylogeny of IPCT/DIPPS revealed an incongruent topology with 16S RNA phylogeny, thus suggesting horizontal gene transfer. The phylogenetic tree of the DIPPS domain was rooted by using phosphatidylinositol phosphate synthase sequences as out-group. The root locates at the separation of genomes with fused and split genes. We propose that the gene encoding DIPPS was recruited from the biosynthesis of phosphatidylinositol. The last DIP-synthesizing ancestor harboured separated genes for IPCT and DIPPS and this architecture was maintained in a crenarchaeal lineage, and transferred by horizontal gene transfer to hyperthermophilic marine Thermotoga species. It is plausible that the driving force for the assembly of those two genes in the early ancestor is related to the acquired advantage of DIP producers to cope with high temperature. This work corroborates the view that Archaea were the first hyperthermophilic organisms.

Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis.

Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo-inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarified but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.