Diphosphoinositol Polyphosphate Phosphohydrolase Research Articles

Using Native and Synthetic Genes to Disrupt Inositol Pyrophosphates and Phosphate Accumulation in Plants.

Inositol pyrophosphates are eukaryotic signaling molecules that have been recently identified as key regulators of plant phosphate sensing and homeostasis. Given the importance of phosphate to current and future agronomic practices, we sought to design plants which could be used to sequester phosphate, as a step in a phytoremediation strategy. To achieve this, we expressed Diadenosine and Diphosphoinositol Polyphosphate Phosphohydrolase (DDP1), a yeast (Saccharomyces cerevisiae) enzyme demonstrated to hydrolyze inositol pyrophosphates, in Arabidopsis thaliana and pennycress (Thlaspi arvense), a spring annual cover crop with emerging importance as a biofuel crop. DDP1 expression in Arabidopsis decreased inositol pyrophosphates, activated Phosphate Starvation Response marker genes, and increased phosphate accumulation. These changes corresponded with alterations in plant growth and sensitivity to exogenously applied phosphate. Pennycress plants expressing DDP1 displayed increases in phosphate accumulation, suggesting that these plants could potentially serve to reclaim phosphate from phosphate-polluted soils. We also identified a native Arabidopsis gene, Nucleoside diphosphate-linked moiety X 13 (NUDIX13), which we show encodes an enzyme homologous to DDP1 with similar substrate specificity. Arabidopsis transgenics overexpressing NUDIX13 had lower inositol pyrophosphate levels and displayed phenotypes similar to DDP1-overexpressing transgenics, while nudix13-1 mutants had increased levels of inositol pyrophosphates. Taken together, our data demonstrates that DDP1 and NUDIX13 can be used in strategies to regulate plant inositol pyrophosphates and could serve as potential targets for engineering plants to reclaim phosphate from polluted environments.

Functional Conservation of the DDP1-type Inositol Pyrophosphate Phosphohydrolases in Land Plant.

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha. We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP7 and 1/3,5-InsP8, highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta. Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha. Collectively, this study provides insights into the regulation of specific PP-InsP messengers by DDP1-type phosphohydrolases in land plants.

New Structural Insights Reveal an Expanded Reaction Cycle for Inositol Pyrophosphate Hydrolysis by Human DIPP1

Nudix hydrolases, an enzyme superfamily with representatives in all classes of organisms, possess a conserved catalytic sequence (Nudix motif), GX5EX7REUXEEXGU, where ‘U’ designates a hydrophobic residue. The Diphosphoinositol Polyphosphate Phosphohydrolases (DIPPs) represent one subgroup of the Nudix family. By metabolizing diphosphoinositol polyphosphates (PP-InsPs), DIPPs switch-off their signaling activities that regulate metabolic homeostasis. However, there has not been a molecular rationalization of DIPPs catalytic mechanism, substrate recognition and reaction specificity. We now provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs (“PCP-InsPs”). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as “suspension cables.” We demonstrate functionality for a triglycine peptide within a β-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity. We have gained considerable structure-based insight into the reaction cycle for DIPP1-catalyzed PP-InsP hydrolysis and can use this information to design inhibitors of an enzyme with proposed roles in obesity, insulin signaling and sarcopenia.

Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping.

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.

Characterizing Enzymes of the Diphosphoinositol Polyphosphate Phosphohydrolase (DIPP) Family.

The diphosphoinositol polyphosphate phosphohydrolases are a subset of the Nudix hydrolase family of enzymes. As such, they metabolize a wide range of substrates, including diphosphoinositol polyphosphates (inositol diphosphates, inositol pyrophosphates), dinucleotide phosphates, nucleosides as well as 5-phosphoribosyl 1-pyrophosphate and inorganic polyphosphate. Here, we describe protocols to optimize these enzymes, with consideration to buffer composition and sample preparation and how to analyze the metabolism of these substrates using high-performance liquid chromatography, giving advice where pitfalls are commonly encountered.

Polyphosphates and Polyphosphatase Activity in the Yeast Saccharomyces cerevisiae during Overexpression of the DDP1 Gene.

The effects of overexpression of yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1) having endopolyphosphatase activity on inorganic polyphosphate metabolism in Saccharomyces cerevisiae were studied. The endopolyphosphatase activity in the transformed strain significantly increased compared to the parent strain. This activity was observed with polyphosphates of different chain length, being suppressed by 2mM tripolyphosphate or ATP. The content of acid-soluble and acid-insoluble polyphosphates under DDP1 overexpression decreased by 9 and 28%, respectively. The average chain length of salt-soluble and alkali-soluble fractions did not change in the overexpressing strain, and that of acid-soluble polyphosphate increased under phosphate excess. At the initial stage of polyphosphate recovery after phosphorus starvation, the chain length of the acid-soluble fraction in transformed cells was lower compared to the recipient strain. This observation suggests the complex nature of DDP1 involvement in the regulation of polyphosphate content and chain length in yeasts.

The enzymes of human diphosphoinositol polyphosphate metabolism.

Diphospho-myo-inositol polyphosphates have many roles to play, including roles in apoptosis, vesicle trafficking, the response of cells to stress, the regulation of telomere length and DNA damage repair, and inhibition of the cyclin-dependent kinase Pho85 system that monitors phosphate levels. This review focuses on the three classes of enzymes involved in the metabolism of these compounds: inositol hexakisphosphate kinases, inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinases and diphosphoinositol polyphosphate phosphohydrolases. However, these enzymes have roles beyond being mere catalysts, and their interactions with other proteins have cellular consequences. Through their interactions, the three inositol hexakisphosphate kinases have roles in exocytosis, diabetes, the response to infection, and apoptosis. The two inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinases influence the cellular response to phosphatidylinositol (3,4,5)-trisphosphate and the migration of pleckstrin homology domain-containing proteins to the plasma membrane. The five diphosphoinositol polyphosphate phosphohydrolases interact with ribosomal proteins and transcription factors, as well as proteins involved in membrane trafficking, exocytosis, ubiquitination and the proteasomal degradation of target proteins. Possible directions for future research aiming to determine the roles of these enzymes are highlighted.

Structural insight into inositol pyrophosphate turnover

The diphosphoinositol polyphosphates ("inositol pyrophosphates"; PP-InsPs) regulate many cellular processes in eukaryotes, including stress responses, apoptosis, vesicle trafficking, cytoskeletal dynamics, exocytosis, telomere maintenance, insulin signaling and neutrophil activation. Thus, the enzymes that control the metabolism of the PP-InsPs serve important cell signaling roles. In order to fully characterize how these enzymes are regulated, we need to determine the atomic-level architecture of their active sites. Only then can we fully appreciate reaction mechanisms and their modes of regulation. In this review, we summarize published information obtained from the structural analysis of a human diphosphoinositol polyphosphate phosphohydrolase (DIPP), and a human diphosphoinositol polyphosphate kinase (PPIP5K). This work includes the analysis of crystal complexes with substrates, products, transition state analogs, and a novel phosphonoacetate substrate analog.

Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate ( K m = 11 μM, k cat = 0.79 s −1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP 5, K m = 0.07 μM, k cat = 0.024 s −1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg 2+ (10–20 mM) or Mn 2+ (1 mM) were preferred for nucleotide hydrolysis and Mg 2+ (0.5–1 mM) or Co 2+ (1–100 μM) for PP-InsP 5 hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H 2O 2 and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP 5 to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP 5 found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP 5 during development.

Sexy gene conversions: locating gene conversions on the X-chromosome

Gene conversion can have a profound impact on both the short- and long-term evolution of genes and genomes. Here, we examined the gene families that are located on the X-chromosomes of human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus) and rat (Rattus norvegicus) for evidence of gene conversion. We identified seven gene families (WD repeat protein family, Ferritin Heavy Chain family, RAS-related Protein RAB-40 family, Diphosphoinositol polyphosphate phosphohydrolase family, Transcription Elongation Factor A family, LDOC1-related family, Zinc Finger Protein ZIC, and GLI family) that show evidence of gene conversion. Through phylogenetic analyses and synteny evidence, we show that gene conversion has played an important role in the evolution of these gene families and that gene conversion has occurred independently in both primates and rodents. Comparing the results with those of two gene conversion prediction programs (GENECONV and Partimatrix), we found that both GENECONV and Partimatrix have very high false negative rates (i.e. failed to predict gene conversions), which leads to many undetected gene conversions. The combination of phylogenetic analyses with physical synteny evidence exhibits high resolution in the detection of gene conversions.

Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway

Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway

Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway

Signals from several receptor tyrosine kinases are transduced by activation of the Ras family of GTP-binding proteins. Activation of Ras initiates a kinase cascade that culminates in activation of the mitogen-activated protein kinases (MAPKs). The MAPKs include the c-jun NH 2-terminal protein kinases (JNKs) and extracellular signal-regulated kinases (ERKs), both of which phosphorylate Elk-1/TCF, a factor that activates transcription of the c- fos gene. In this report, we identify a novel 19 kDa gene product as a negative regulator of signaling through the ERK1/2 pathway. While these studies were in progress, the human homologue of this gene was characterized as diphosphoinositol polyphosphate phosphohydrolase (DIPP1) [EMBO J. 17 (1998) 6599], a phosphohydrolase that converts diphosphate groups on diphosphoinositol polyphosphates to monophosphates. Ectopic expression of murine DIPP1 (muDIPP1) blocked activation of the c- fos promoter by the ERK1/2 pathway. Inhibition of signal transduction through the ERK1/2 pathway by muDIPP1 occurs at or downstream from activation of MEK. In vitro kinase studies suggest that muDIPP1 is not a direct inhibitor of MEK or ERK activity, although, ectopic expression at near physiological levels results in attenuation of ERK phosphorylation in vivo. Interestingly, a site mutant of muDIPP1 lacking phosphohydrolase activity blocked signaling through the ERK1/2 pathway with greater efficiency than wild-type muDIPP1. This result suggests that inhibition of signaling through the ERK1/2 pathway is a distinct function of muDIPP1 that is not dependent on, but may be regulated by, its activity as a phosphohydrolase.

Paralogous murine Nudt10 and Nudt11 genes have differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases.

We previously described paralogous human genes [NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety 'X')-type motif, also known as the 'nudix'-type motif]] encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem. 277, 32730-32738]. Normally, gene duplication is redundant, and lacks biological significance. Is this true for the DIPP3 genes? We address this question by characterizing highly-conserved murine Nudt10 and Nudt11 homologues of the human genes. Thus these genes must have been duplicated prior to the divergence of primates and sciurognath rodents, approx. 115 million years ago, greatly exceeding the 4 million year half-life for inactivation of redundant paralogues; our data therefore indicate that the DIPP3 duplication is unusual in being physiologically significant. One possible functional consequence is gene neofunctionalization, but we exclude that, since Nudt10 and Nudt11 encode identical proteins. Another possibility is gene subfunctionalization, which we studied by conducting the first quantitative expression analysis of these genes. We demonstrated high Nudt10 expression in liver, kidney and testis; Nudt11 expression is primarily restricted to the brain. This differential, but complementary, expression pattern indicates that subfunctionalization is the evolutionary consequence of DIPP3 gene duplication. Our kinetic data argue that diphosphoinositol polyphosphates are more physiologically relevant substrates for DIPP3 than are either diadenosine hexaphosphate or 5-phosphoribosyl 1-pyrophosphate. Thus the significance of the Nudt10/Nudt11 duplication is specific hydrolysis of diphosphoinositol polyphosphates in a tissue-dependent manner.

Nudix Hydrolases That Degrade Dinucleoside and Diphosphoinositol Polyphosphates Also Have 5-Phosphoribosyl 1-Pyrophosphate (PRPP) Pyrophosphatase Activity That Generates the Glycolytic Activator Ribose 1,5-Bisphosphate

A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.

An Adjacent Pair of Human NUDT Genes on Chromosome X Are Preferentially Expressed in Testis and Encode Two New Isoforms of Diphosphoinositol Polyphosphate Phosphohydrolase

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k(cat)/K(m) = 0.2 x 10(5) m(-1) s(-1)) compared with diphosphoinositol polyphosphates (k(cat)/K(m) = 2-40 x 10(5) m(-1) s(-1)). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40-50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3alpha; NUDT11 encodes hDIPP3beta) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates.

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.

Molecular Cloning of a Novel Isoform of Diphosphoinositol Polyphosphate Phosphohydrolase:: A Potential Target of Lithium Therapy

The mechanisms underlying the therapeutic effects of lithium are largely unknown but may involve progressive adaptive alterations at the level of gene expression. Using differential display PCR, we identify a novel cDNA fragment, the expression of which was increased in the rat frontal cortex after 5 weeks of lithium administration. A full-length cDNA (2954-nt) was cloned by arrayed cDNA library screening, and sequencing of the clone revealed an open reading frame of 537-bp encoding a 179-residue protein. Amino acid sequence comparisons revealed that our clone is a member of the Nudix hydrolase family, with the highest percentage of homology (95%) being with a subtype of human diphosphoinositol polyphosphate phosphohydrolase, hDIPP2. Northern blot analysis revealed that chronic lithium treatment significantly increased rDIPP2 mRNA levels in frontal cortex, but not in hippocampus, midbrain, and cerebellum. The effect of lithium on rDIPP2 mRNA expression was not shared by two other anticonvulsant mood stabilizers, carbamazepine and valproate. Time-course studies showed that 1-week of lithium had no effect on rDIPP2 mRNA abundance in the frontal cortex. Our results suggest that DIPP2 may represent a biologically relevant target of lithium therapy, further supporting the notion that abnormalities in inositol phosphate metabolism may be significant in the pathophysiology and pharmacotherapy of bipolar disorder.

Genetic rationale for microheterogeneity of human diphosphoinositol polyphosphate phosphohydrolase type 2

Selective expression of enzymes that adjust the intensity of turnover of diphosphoinositolpolyphosphates may regulate vesicle trafficking and DNA repair. For example, the type 2 human diphosphoinositolpolyphosphate phosphohydrolases (hDIPP2α and 2β) are distinguished by a solitary amino-acid residue; the type 2β isoform contains Gln86 whereas the type 2α isoform does not, yet the latter has 2–5 fold more catalytic activity than its β counterpart (J. Biol.Chem. (2000) 12730). We discovered that both α and β-type mRNAs were co-expressed in clonal cell-lines. We sought a genetic explanation for this microheterogeneity. Two BACs containing distinct, but intronless, hDIPP2β genes were cloned. Only one of these genes could potentially give rise to our previously characterized hDIPP2β mRNA; the other gene has several sequence differences and, in any case, is likely a processed pseudogene. These BACS were mapped to 1q12-q21 and 1p12-p13 by FISH. No analogous intronless hDIPP2α gene was detected by analysis of 21 individual genomic DNAs. However, sequence analysis of a third hDIPP2 gene (at 12q21) places the Gln86 CAG codon within an AGCAG pentamer, offering adjacent, alternate intronic 3′-boundaries. Thus, ‘intron boundary skidding’ by spliceosomes provides a mechanism for yielding both hDIPP2α and hDIPP2β mRNAs. Our studies expand the repertoire of molecular mechanisms regulating diphosphoinositolpolyphosphate metabolism and function.

The Inositol Hexakisphosphate Kinase Family: CATALYTIC FLEXIBILITY AND FUNCTION IN YEAST VACUOLE BIOGENESIS

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

Discovery of Molecular and Catalytic Diversity among Human Diphosphoinositol-Polyphosphate Phosphohydrolases: AN EXPANDING NUDT FAMILY

The turnover of the "high energy" diphosphoinositol polyphosphates by Ca(2+)- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype human diphosphoinositol-polyphosphate phosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2alpha and hDIPP2beta, distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Gln(86)). Candidate DIPP2alpha and DIPP2beta homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2alpha > hDIPP2beta. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX(2)GX(6)G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3'-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.