Cytidylyltransferase Activity Research Articles

Deciphering structure, function and mechanism of Plasmodium IspD homologs from their evolutionary imprints.

Malaria is a life-threatening mosquito-borne blood disease caused by infection with Plasmodium parasites. Anti-malarial drug resistance is a global threat to control and eliminate malaria and therefore, it is very important to discover and evaluate new drug targets. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) homolog is a second in vivo target for fosmidomycin within isoprenoid biosynthesis in malarial parasites. In the present study, we have deciphered the sequence-structure-function integrity of IspD homologs based on their evolutionary imprints. The function and catalytic mechanism of them were also intensively studied by using sequence-structure homology, molecular modeling, and docking approach. Results of our study indicated that substrate-binding and dimer interface motifs in their structures were extensively conserved and part of them closely related to eubacterial origins. Amino acid substitutions in their coiled-coil regions found to bring a radical change in secondary structural elements, which in turn may change the local structural environment. Arg or Asp was identified as a catalytic site in plasmodium IspD homologs, contributing a direct role in the cytidylyltransferase activity similar to bacterial IspD. Results of molecular docking studies demonstrated how anti-malarial drugs such as fosmidomycin and FR-900098 have competitively interacted with the substrate-binding site of these homologs. As shown by our analysis, species-specific evolutionary imprints in these homologs determine the sequence-structure-function-virulence integrity and binding site alterations in order to confer anti-malarial drug resistance.

1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50nM of lovastatin for 60h, lovastatin was found to decrease the incorporation of [3H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [3H]sphingomyelin by lovastatin. The increase of [3H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.

Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase

Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE Regulates Phosphatidylcholine Biosynthesis in Arabidopsis by Phosphatidic Acid-Mediated Activation of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Activity.

Regulation of membrane lipid biosynthesis is critical for cell function. We previously reported that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 (PAH1) and PAH2 stimulates net phosphatidylcholine (PC) biosynthesis and proliferation of the endoplasmic reticulum (ER) in Arabidopsis thaliana. Here, we show that this response is caused specifically by a reduction in the catalytic activity of the protein and positively correlates with an accumulation of its substrate, phosphatidic acid (PA). The accumulation of PC in pah1 pah2 is suppressed by disruption of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE1 (CCT1), which encodes a key enzyme in the nucleotide pathway for PC biosynthesis. The activity of recombinant CCT1 is stimulated by lipid vesicles containing PA. Truncation of CCT1, to remove the predicted C-terminal amphipathic lipid binding domain, produced a constitutively active enzyme. Overexpression of native CCT1 in Arabidopsis has no significant effect on PC biosynthesis or ER morphology, but overexpression of the truncated constitutively active version largely replicates the pah1 pah2 phenotype. Our data establish that membrane homeostasis is regulated by lipid composition in Arabidopsis and reveal a mechanism through which the abundance of PA, mediated by PAH activity, modulates CCT activity to govern PC content.

Mitochondrial Phosphatidylethanolamine Level Modulates Cyt c Oxidase Activity to Maintain Respiration Capacity in Arabidopsis thaliana Rosette Leaves

Phosphatidylethanolamine is the predominant phospholipid of the mitochondrial inner membrane. In Arabidopsis, pect1-4 mutants exhibit reduced cellular phosphatidylethanolamine levels owing to reduced CTP:phosphorylethanolamine cytidylyltransferase (PECT; EC 2.7.7.14) activity. Consequently, pect1-4 mutants may have decreased mitochondrial phosphatidylethanolamine levels, thereby affecting respiration capacity. Wild-type and pect1-4 plants grew similarly under a short-day condition until 5 weeks, when pect1-4 leaves had slightly less Chl. Total respiration was comparable between wild-type and pect1-4 leaves at 3 weeks and then increased 2-fold in the wild-type but only 1.1-fold in pect1-4 leaves. Compared with the wild type, the Cyt oxidase pathway capacity was reduced by 36% in pect1-4 leaves at 5 weeks and by 43% in pect1-4 mitochondria in 5-week-old rosette leaves. Maximal Cyt c oxidase (COX) activity was 20% lower in pect1-4 mitochondria than in wild-type mitochondria at 5 weeks despite comparable COX II protein levels in mitochondria at that time. Furthermore, COX II protein levels doubled in both wild-type and pect1-4 mitochondria between 3 and 5 weeks. Phosphatidylethanolamine levels were similar between mitochondria from these plants at 3 weeks and then increased by 6.4% in wild-type mitochondria and decreased by 6.5% in pect1-4 mitochondria by 5 weeks. Phosphatidylcholine levels compensated for the decreases in phosphatidylethanolamine levels. COX activity was lower in pect1-4 mitochondria at 5 weeks, most probably due to reduced phosphatidylethanolamine levels and/or an altered phosphatidylethanolamine:phosphatidylcholine ratio. Thus, PECT1 regulates mitochondrial phosphatidylethanolamine levels, which are important for maintaining respiration capacity in Arabidopsis leaves during prolonged growth under short-day conditions.

HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using 13C MRS to monitor [1,2-13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by 31P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using 1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

BackgroundThe gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.ResultsHere, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.ConclusionsTaken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di- myo -Inositol-Phosphate Synthesis in (Hyper)Thermophiles

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.

Imaging Lipid Synthesis in Hepatocellular Carcinoma with [Methyl-11C]Choline: Correlation with In Vivo Metabolic Studies

PET with [methyl-(11)C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG-avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-(14)C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio-high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89±0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5'-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

Dietary intake of S-(alpha-carboxybutyl)-DL-homocysteine induces hyperhomocysteinemia in rats.

Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(alpha-carboxybutyl)-DL-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed in the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an L-amino acid-defined diet either containing or devoid of CBHcy (5 mg per meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine beta-synthase activity and immunodetectable protein decreased (56% and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats, and S-adenosylhomocysteine levels did not change. Furthermore, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology, and plasma markers of liver damage. We conclude that CBHcy-mediated BHMT inhibition causes an elevation in total plasma Hcy that is not normalized by the folate-dependent conversion of Hcy to methionine. Furthermore, metabolic changes caused by BHMT inhibition affect cystathionine beta-synthase and glycine N-methyltransferase activities, which further deteriorate plasma Hcy levels.

Nuclear export of the rate-limiting enzyme in phosphatidylcholine synthesis is mediated by its membrane binding domain

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), the rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis, is activated by translocation to nuclear membranes. However, CCTalpha is cytoplasmic in cells with increased capacity for PtdCho synthesis and following acute activation, suggesting that nuclear export is linked to activation. The objective of this study was to identify which CCTalpha domains were involved in nuclear export in response to the lipid activators farnesol (FOH) and oleate. Imaging of CCT-green fluorescent protein (GFP) mutants expressed in CCTalpha-deficient CHO58 cells showed that FOH-mediated translocation to nuclear membranes and export to the cytoplasm required the membrane binding amphipathic helix (domain M). Nuclear export was reduced by a mutation that mimics constitutive phosphorylation of the CCT phosphorylation (P) domain. However, domain M alone was sufficient to promote translocation to the nuclear envelope and export of a nuclear-localized GFP construct in FOH- or oleate-treated CHO58 cells. In the context of acute activation with lipid mediators, nuclear export of CCT-GFP mutants correlated with in vitro activity but not PtdCho synthesis. This study describes a nuclear export pathway that is dependent on membrane interaction of an amphipathic helix, thus linking lipid-dependent activation to the nuclear/cytoplasmic distribution of CCTalpha.

Why does a defect in choline kinase beta cause muscular dystrophy in mice?

Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles. There is no specific treatment to stop or reverse any form of MD due to limited knowledge. We recently uncovered a natural mutation (rmd) in the Chkb gene that resulted in a complete loss of choline kinase (CK) activity in skeletal muscle, and the mice developed severe and progressive hindlimb MD. It is the first demonstration of a defect in a phospholipid biosynthetic enzyme causing MD. Why does muscular dystrophy occur? We observed decreased phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio both in hindlimb muscle and in mitochondria associated with sarcolemmal integrity and megamitochondria formation. Decreased PC level is due to three factors: down‐regulated CTP:phosphocholine cytidylyltransferase (CT) activity, the rate‐limiting enzyme for PC biosynthesis; increased PC turnover due to increased phospholipase activity, and a limited source of phosphocholine in Chkb−/– mice. In vivo experiments further demonstrated that the incorporation ability of 3H‐choline into PC was significantly decreased in affected muscle. Decreased regeneration ability of Chkb−/–mice may also contribute to muscular dystrophy. In affected muscle there was increased expression of myostatin and decreased proliferating cell nuclear antigen (PCNA).This research was supported by a grant from Alberta Heritage Foundation for Medical Research and Canadian Institutes of Health Research.

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.

Di-myo-inositol 1,1'-phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-mono-phosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418-TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. (31)P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipB-encoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.

Coordinate Regulation of Phospholipid Biosynthesis and Secretory Pathway Gene Expression in XBP-1(S)-induced Endoplasmic Reticulum Biogenesis

Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein response-mediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDP-choline pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis.

Metabolic Profiling of Glycerophospholipid Synthesis in Fibroblasts Loaded with Free Cholesterol and Modified Low Density Lipoproteins

Currently, the detailed regulation of major pathways of glycerophospholipid synthesis upon cholesterol loading is largely unknown. Therefore, a detailed lipid metabolic profiling using stable isotope-labeled choline, ethanolamine, and serine was performed by quantitative electrospray ionization tandem mass spectrometry (ESI-MS/MS) in free cholesterol (FC), oxidized (Ox-LDL) and enzymatically modified LDL (E-LDL)-loaded primary human skin fibroblasts. As previously described, an adaptive induction of phosphatidylcholine (PC) synthesis via CDP-choline was found upon FC loading. In contrast to PC, CDP-ethanolamine-mediated phosphatidylethanolamine (PE) synthesis was inhibited by FC incubation. Furthermore, FC induced a shift toward polyunsaturated PE and PC species, which was mediated primarily by PE biosynthesis but not PE remodeling, whereas PC species were shifted mainly by fatty acid (FA) remodeling of existing PC. Modified lipoprotein incubation revealed rather different effects on glycerophospholipid synthesis. E-LDL greatly enhanced PC synthesis, whereas Ox-LDL did not change PC synthesis. Addition of different free FAs (FFA) with and without FC coincubation, as major components of E-LDL, clearly indicated an incorporation of FFA into newly synthesized PC and PE species as well as FFA as important driving force for PC synthesis. Because FC and FFA are known to affect lipid membrane properties including membrane curvature, these data support that CTP:phosphocholine cytidylyl-transferase activity and consequently PC synthesis are regulated by modulation of membrane characteristics at the cellular level. In conclusion, the application of high throughput metabolic profiling of major glycerophospholipid pathways by ESI-MS/MS is a powerful tool to unravel mechanisms underlying the regulation of cellular lipid metabolism.

Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption

A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.