2-Aminoethylarsonic acid as an analogue of ethanolamine phosphate. Endowment of ethanolamine-phosphate cytidylyltransferase with CTP pyrophosphatase activity.
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ABSTRACT:
SUBMITTER: Visedo-Gonzalez E
PROVIDER: S-EPMC1138663 | biostudies-other | 1989 May
REPOSITORIES: biostudies-other
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Project description:Several possible control mechanisms for CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity in pea (Pisum sativum L.) stems were investigated. Indol-3-ylacetic acid (IAA) treatment of the pea stems decreased total cytidylyltransferase activity but did not affect its subcellular distribution. Oleate (2 mM) caused some stimulation of enzyme activity by release of activity from the microsomal fraction into the cytosol, but neither phosphatidylglycerol nor monoacyl phosphatidylethanolamine had an effect on activity or subcellular distribution. A decrease in soluble cytidylyltransferase protein concentrations was found in IAA-treated pea stems, but this was not sufficient to account for all of the decrease in cytidylyltransferase activity. A 50% inhibition of enzyme activity could be obtained with 0.2 mM-CMP, which indicated possible allosteric regulation. Similar inhibition was obtained with 1.5 mM-ATP, but other nucleotides had no effect. The cytidylyltransferase enzyme protein was not directly phosphorylated, and the inhibition with 1.5 mM-ATP occurred with the purified enzyme, thus excluding an obligatory mediation via a modulator protein. The results indicate that the cytosolic form of cytidylyltransferase is the most important in pea stem tissue and that the decrease in cytidylyltransferase activity in IAA-treated material appears to be brought about by several methods.
| S-EPMC1147495 | biostudies-other
Project description:Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3'-phosphate-1'-phosphate from CDP-inositol and L-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K(m) values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V(max) determined at 90°C was 62.9 μmol · min(-1) · mg of protein(-1). The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X(4)-P-K. CTP, L-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.
| S-EPMC3133074 | biostudies-literature
Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.
| S-EPMC1951816 | biostudies-literature
Project description:We examined the effects of the bioactive lipid, sphingosine, on the expression of the rate-limiting enzyme involved in surfactant phosphatidylcholine synthesis, CCTalpha (CTP:phosphocholine cytidylyltransferase alpha). Sphingosine decreased phosphatidylcholine synthesis by inhibiting CCT activity in primary alveolar type II epithelia. Sphingosine decreased CCTalpha protein and mRNA levels by approx. 50% compared with control. The bioactive lipid did not alter CCTalpha mRNA stability, but significantly inhibited its transcriptional rate. In murine lung epithelia, sphingosine selectively reduced CCTalpha promoter-reporter activity when transfected with a 2 kb CCTalpha promoter/luciferase gene construct. Sphingosine also decreased transgene expression in murine type II epithelia isolated from CCTalpha promoter-reporter transgenic mice harbouring this 2 kb proximal 5'-flanking sequence. Deletional analysis revealed that sphingosine responsiveness was mapped to a negative regulatory element contained within 814 bp upstream of the coding region. The results indicate that bioactive sphingolipid metabolites suppress surfactant lipid synthesis by inhibiting gene transcription of a key surfactant biosynthetic enzyme.
| S-EPMC1133833 | biostudies-literature
Project description:The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha.
| S-EPMC2174170 | biostudies-literature
Project description:Phosphatidylethanolamine (PE) is the most abundant inner membrane phospholipid. PE synthesis from ethanolamine and diacylglycerol is regulated primarily by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2(+/-) mice have reduced PE synthesis and, as a consequence, perturbed glucose and fatty acid metabolism, which gradually leads to the development of hyperlipidemia, obesity, and insulin resistance. Glucose and fatty acid uptake and the corresponding transporters Glut4 and Cd36 are similarly impaired in male and female Pcyt2(+/-) hearts. These mice also have similarly reduced phosphatidylinositol 3-kinase (PI3K)/Akt1 signaling and increased reactive oxygen species (ROS) production in the heart. However, only Pcyt2(+/-) males develop hypertension and cardiac hypertrophy. Pcyt2(+/-) males have upregulated heart AceI expression, heart phospholipids enriched in arachidonic acid and other n-6 polyunsaturated fatty acids, and dramatically increased ROS production in the aorta. In contrast, Pcyt2(+/-) females have unmodified heart phospholipids but have reduced heart triglyceride levels and altered expression of the structural genes Acta (low) and Myh7 (high). These changes together protect Pcyt2(+/-) females from cardiac dysfunction under conditions of reduced glucose and fatty acid uptake and heart insulin resistance. Our data identify Pcyt2 and membrane PE biogenesis as important determinants of gender-specific differences in cardiac lipids and heart function.
| S-EPMC4524120 | biostudies-literature
Project description:Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.
| S-EPMC1152942 | biostudies-other
Project description:CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation.
| S-EPMC1133914 | biostudies-literature
Project description:Phosphatidylethanolamine (PE) is the most abundant lipid on the protoplasmatic leaflet of cellular membranes. It has a pivotal role in cellular processes such as membrane fusion, cell cycle regulation, autophagy, and apoptosis. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme in de novo biosynthesis of PE from ethanolamine and diacylglycerol by the CDP-ethanolamine Kennedy pathway. The following is a summary of the current state of knowledge on Pcyt2 and how splicing and isoform specific differences could lead to variations in functional properties in this family of enzymes. Results from the most recent studies on Pcyt2 transcriptional regulation, promoter function, autophagy, and cell growth regulation are highlighted. Recent data obtained from Pcyt2 knockout mouse models is also presented, demonstrating the essentiality of this gene in embryonic development as well as the major physiological consequences of deletion of one Pcyt2 allele. Those include development of symptoms of the metabolic syndrome such as elevated lipogenesis and lipoprotein secretion, hypertriglyceridemia, liver steatosis, obesity, and insulin resistance. The objective of this review is to elucidate the nature of Pcyt2 regulation by linking its catalytic function with the regulation of lipid and energy homeostasis.
| S-EPMC3588000 | biostudies-literature
Project description:CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50-90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.
| S-EPMC3979399 | biostudies-literature