Plant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

myo-Inositol monophosphatase (IMP) is a soluble, Li(+)-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose 6-phosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LeIMP) and show that the plant enzyme is encoded by a small gene family. Three different LeIMP cDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LeIMPs are inhibited by low concentrations of LiCl. LeIMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. Immunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LeIMPs encoded by the genes. Immunoreactive proteins in the same size range are also present in several other plants. Immunolocalization studies indicated that many cell types within seedlings accumulate LeIMP proteins. In particular, cells associated with the vasculature express high levels of LeIMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants.     

Yeast inositol mono- and trisphosphate levels are modulated by inositol monophosphatase activity and nutrients

Yeast lithium-sensitive inositol monophosphatase (IMPase) is encoded by a non-essential gene pair (IMP1 and IMP2). Inhibition of IMPase with either Li(+) or Na(+) or a double null mutation imp1 imp2 causes increased levels of inositol monophosphates and reduced level of inositol 1,4,5-trisphosphate. Overexpression of the IMP2 gene has the opposite effects and these results suggest that IMPase activity is limiting for the inositol cycle. Addition of ammonium to cells starved for this nutrient results in a decrease of inositol monophosphates and an increase of inositol 1,4,5-triphosphate, pointing to simultaneous regulation of both inositol 1,4,5-triphosphate production and IMPase activity.     

Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

Background  

Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos"), that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE) DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP) form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates.     

Genoprotective pathways. II. Attenuation of oxidative DNA damage by isopentenyl diphosphate

Oxidative stress is believed to play a role in the pathogenesis of many diseases. Here we report that isopentenyl diphosphate (IPP), the 5-carbon building unit of all isoprenoids, is a potent antioxidant that is capable of inhibiting oxidative DNA damage at picomolar concentrations (IC50 = 1.7 x 10(-11) M). The diphosphate moiety is essential, since isopentenyl monophosphate (IMP) is unable to trigger antioxidative signaling. The 20-carbon isoprenyl, geranylgeranyl diphosphate (GGPP), but not the 15-carbon farnesyl diphosphate, displays similar genoprotective effects. The pathway activated by IPP is distinct from that of 2-chloroadenosine (2CA). 2CA-mediated genoprotective signaling is transduced through an A2a or A2b adenosine receptor (AR) and can be blocked by the cyclic AMP (cAMP)-dependent protein kinase (PKA) inhibitor, H-89. In contrast, IPP signaling is independent of A2aAR, A2bAR, cAMP or PKA. Unlike the 2CA-mediated pathway, the effect of IPP is dependent on the mevalonate pathway, a geranylgeranylated protein and on intact proteasome activity. Thus, IPP is a potent activator of a novel genoprotective pathway. These findings shed new light on the role of isoprenoids in oxidative stress biology and may help to develop novel preventive strategies against oxidative damage.     

A secreted salivary inositol polyphosphate 5-phosphatase from a blood-feeding insect: allosteric activation by soluble phosphoinositides and phosphatidylserine

Type II inositol polyphosphate 5-phosphatases (IPPs) act on both soluble inositol phosphate and phosphoinositide substrates. In many cases, these enzymes occur as multidomain proteins in which the IPP domain is linked to lipid-binding or additional catalytic domains. Rhodnius prolixus IPPRp exists as an isolated IPP domain which is secreted into the saliva of this blood-feeding insect. It shows selectivity for soluble and lipid substrates having a 1,4,5-trisphosphate substitution pattern while only poorly hydrolyzing substrates containing a D3 phosphate. With soluble diC8 PI(4,5)P(2) as a substrate, sigmoidal kinetics were observed, suggesting the presence of allosteric activation sites. Surprisingly, IPPRp-mediated hydrolysis of PI(4,5)P(2) and PI(3,4,5)P(3) was also stimulated up to 100-fold by diC8 PI(4)P and diC8 phosphatidylserine (PS). The activation kinetics were again sigmoidal, demonstrating that the allosteric sites recognize nonsubstrate phospholipids. Activation was positively cooperative, and analysis by the Hill equation suggests that at least three to four allosteric sites are present. In a vesicular system, hydrolysis of PI(4,5)P(2) followed a surface dilution kinetic model, and as expected, PS was found to be strongly stimulatory. If allosteric activation of type II IPPs by PI(4)P and PS is a widespread feature of the group, it may represent a novel regulatory mechanism for these important enzymes.     

Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway

Phytic acid, myo-inositol-hexakisphosphate (InsP(6)), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1D-myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P(3)) is sequentially phosphorylated to InsP(6). We identified 12 genes from rice (Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1D-myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers.     

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. We address the role of ZBP-1/VERA through analysis of the Drosophila melanogaster homologue insulin growth factor II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, we identified the IMP-binding element (IBE) UUUAY, a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. Our findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis.     

The inositol pyrophosphate pathway in health and diseases

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate‐containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet‐induced obesity (DIO), type‐2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio‐metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.     

Reciprocal interaction of hemoglobin with oxygen and protons. The influence of allosteric polyanions.

The interaction of three inositol esters, inositol hexaphosphate (IHP), inositol pentaphosphate (IPP), and inositol hexasulfate (IHS), with hemoglobin has been investigated. The proton uptake method was used to obtain the six binding constants for deoxy- and oxyhemoglobin. These data combined with oxygen binding curves over a range of cofactor concentrations were used to test theoretical and empirical equations relating the affinity of hemoglobin for oxygen and allosteric effectors. The Bohr and Haldane coefficients in the presence of the inositol esters are unequal at low, but not at high, concentration of the cofactors. The maximum value reached by both parameters increases with the number of negative charges of the polyanion. 2,3-Diphosphoglycerate (DPG) differs sharply from the inositol esters since even at high concentrations of this cofactor, the Haldane coefficient remains elevated. This is a reflection of the negligible affinity of DPG for fully oxygenated hemoglobin.     

Mutational Analysis of the RecJ Exonuclease of Escherichia coli: Identification of Phosphoesterase Motifs

The recJ gene, identified in Escherichia coli, encodes a Mg(+2)-dependent 5'-to-3' exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including Bacillus and Helicobacter, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of E. coli RecJ. These motifs may define interactions with Mg(2+) ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of Mycoplasma, a domain of putative polyA polymerases in Synechocystis and Aquifex, PRUNE of Drosophila, and an exopolyphosphatase (PPX1) of Saccharomyces cereviseae. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.     

Intramembrane particles and the organization of lymphocyte membrane proteins

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- β-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.     

Actions of insulin, epinephrine, and dibutyryl cyclic adenosine 5'-monophosphate on fat cell protein phosphorylations. Cyclic adenosine 5'-monophosphate dependent and independent mechanisms.

Endogenous and hormone-induced protein (polypeptide) phosphorylations were studied in isolated rat fat cells, in fat pads, and in subcellular fractions obtained from fat tissue under different physiological conditions. Insulin (25-100 muU/ml) increased the incorporation of 32P into two proteins: insulin-phosphorylated proteins (IPP 140 and IPP 50; similar to 140,000 and 50,000 daltons, respectively). Epinephrine (10(-7)-10(-6) M) increased the incorporation of 32P into another protein: epinephrine-phosphorylated protein (EPP 60-65; similar to 60,000-65,000 daltons). Endogenous IPP 140 phosphorylation in fat cells obtained from fasted and refed rats was similar to that of insulin in normal cells. Studies of insulin and epinephrine interactions showed that insulin increased IPP 140 phosphorylation even in the presence of epinephrine or lithium (25 mM times 10(-3) M). dibutyryl cyclic AMP (5 times 10(-4) M) markedly stimulated EPP 60-65 phosphorylation, but neither epinephrine (10(-7)-10(-6) M) nor dibutyryl cyclic AMP reproduced insulin's phosphorylation of APP 140. Lithium inhibited both endogenous and epinephrine-stimulate EPP 60-65 phosphorylation, but did not inhibit that induced by dibutyryl cyclic AMP. These findings suggest that insulin stimulated a specific, cyclic AMP independent protein kinase for IPP 140 phosphorylation. Cell-free extracts from insulin-treated fat tissue catalyzed the specific transfer of 32P from ATP to IPP 140 more rapidly than control extracts. No differences in the total receptor protein or total protein kinase activity using [gamma(-32P]ATP were noted between insulin-treated and control preparations. IPP 140 may be either (a) an insulin-sensitive protein kinase (phosphotransferase) or (b) a protein whose function is regulated by an insulin-sensitive protein kinase or phosphatase.     

A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development.     

Effects of acclimation to altitude on oxygen affinity and organic phosphate concentrations in pigeon blood.

Hematocrit ratio, hemoglobin concentration and blood oxygen affinity, Bohr effect factor and Hill coefficient, adenosine triphosphate and inositol pentaphosphate (IPP) concentrations were studied in blood of adult pigeons exposed first at 140 m, and then for 3 weeks at 4000 m in an altitude chamber. At altitude, the hematocrit ratio and hemoglobin concentration significantly increased, IPP concentration decreased, and P₅₀ did not change. A lower mean red cell age and a higher hemoglobin concentration may account for the unchanged P₅₀. Adaptation to hypoxia of the tissue oxygen supply was shown by a greater blood O₂ capacitance (ΔC_HbO₂/Δ_o2) in the physiological range of oxygen partial pressures.     

The interaction of inositol pentaphosphate with the hemoglobins of highland and lowland geese.

We have studied the binding of inositol pentaphosphate (IPP) to the hemoglobins from two species of goose living at low and high altitudes, using the proton absorption method. Measurements were done at 25 and 37 degrees C in a pH range between 6.0 and 8.8. The bird hemoglobins show a high affinity and a binding stoichiometry of 1 IPP molecule/hemoglobin tetramer both in the ligated and unligated state, indicating the same binding site for IPP in oxy- and deoxyhemoglobin. The results indicate that the interaction of IPP with both geese hemoglobins is very similar. For the deoxyhemoglobins of both species the IPP-binding constant shows a strong pH dependence extending over a wide pH range (i.e. +/- 2 x 10(6) M at pH 8.8 and +/- 6 x 10(10) M at pH 6.0). The binding constant of IPP for the oxyhemoglobins shows a much weaker pH dependence (i.e. +/- 4 x 10(4) M at pH 8.8 and +/- 3 x 10(6) M at pH 6.0), indicating that the interaction of IPP with the goose hemoglobin is strongly dependent on the state of ligation of the protein. The IPP binding constants for the oxy- and deoxyhemoglobins are found to be in good agreement with the IPP-induced change in oxygen affinity of both hemoglobins as estimated from oxygen binding curves.     

Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA

Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single alpha-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a beta-sheet subdomain and an alpha-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The beta-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP.     

An Integrative Approach to the Study of Filamentous Oligomeric Assemblies,with Application to RecA

Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called “extended” and “compressed” forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.     

Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.

Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an important target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E. coli IMP dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.     

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.