Co-ordinate regulation of ethanolamine kinase and phosphoethanolamine cytidylyltransferase in the biosynthesis of phosphatidylethanolamine in rat liver.
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ABSTRACT:
SUBMITTER: Infante JP
PROVIDER: S-EPMC1186686 | biostudies-other | 1979 Jun
REPOSITORIES: biostudies-other
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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, -? and -?; 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 Pcyt2? and -? 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. Pcyt2? was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2? was phosphorylated within the ?-specific motif that is spliced out in Pcyt2? and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2? 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 Pcyt2? was phosphorylated by PKC?, PKC?I, and PKC?II. Pcyt2? Ser-215 was also directly phosphorylated with PKC?. Mapping of the Pcyt2?- and -?-phosphorylated sites to the solved structure of a human Pcyt2? 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
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:The effect of partial (70%) hepatectomy on phosphatidylethanolamine (PE) synthesis was studied in rat liver during the first 4 post-operative days. Between 4 and 96 h after partial hepatectomy, the mass of PE increased from 30% to 80% of sham-operation values. In line with the increase in PE mass, the rate of PE synthesis in vivo from [14C]ethanolamine was stimulated 1.6- and 1.3-fold at 22 and 48 h after partial hepatectomy respectively. Surprisingly, the activity of CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) was virtually unchanged after partial hepatectomy. In addition, neither ethanolamine kinase (EC 2.7.1.82) nor ethanolaminephosphotransferase (EC 2.7.8.1) showed any changes in activity over the time period studied. Hepatic levels of ethanolamine and phosphoethanolamine were drastically increased after partial hepatectomy, as compared with sham operation, whereas levels of CDP-ethanolamine and microsomal diacylglycerol were not affected. Interestingly, partial hepatectomy caused the concentration of free ethanolamine in serum to increase from 29 microM to approx. 50 microM during the first day after surgery. In hepatocytes isolated from non-operated animals, incorporation of [3H]ethanolamine into PE was stimulated by increasing the ethanolamine concentration from 10 up to 50 microM, whereas the radioactivity associated with phosphoethanolamine only increased at ethanolamine concentrations higher than 30 microM. Taken together, our results indicate that the observed increase in serum ethanolamine concentration after partial hepatectomy is probably responsible for both the increase in PE biosynthesis and the accumulation of ethanolamine and phosphoethanolamine in regenerating liver.
| S-EPMC1130992 | biostudies-other
Project description:CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.
| S-EPMC1220530 | biostudies-other
Project description:Sphingomyelin (SM) is a vital component of mammalian membranes, providing mechanical stability and a structural framework for plasma membrane organization. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase in the Golgi lumen. Drosophila lacks SM and instead synthesizes the SM analogue ceramide phosphoethanolamine (CPE) as the principal membrane sphingolipid. The corresponding CPE synthase shares mechanistic features with enzymes mediating phospholipid biosynthesis via the Kennedy pathway. Using a functional cloning strategy, we here identified a CDP-ethanolamine:ceramide ethanolamine phosphotransferase as the enzyme responsible for CPE production in Drosophila. CPE synthase constitutes a new branch within the CDP-alcohol phosphotransferase superfamily with homologues in Arthropoda (insects, spiders, mites, scorpions), Cnidaria (Hydra, sea anemones), and Mollusca (oysters) but not in most other animal phyla. The enzyme resides in the Golgi complex with its active site facing the lumen, contrary to the membrane topology of other CDP-alcohol phosphotransferases. Our findings open up an important new avenue to address the biological role of CPE, an enigmatic membrane constituent of a wide variety of invertebrate and marine organisms.
| S-EPMC3630839 | 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.
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Project description:Two-dimensional gel-electrophoretic analysis combined with fluorography and densitometric quantification was used to examine the effects of glucose on the biosynthesis of rat pancreatic islet proteins. An increase in the medium glucose concentration from 2.8 to 16.7 mM produced a 10-20 fold stimulation in the synthesis of 10 out of 260 detected islet proteins, as judged by incorporation of [35S]methionine during a 20 min incubation. The synthetic rates of the majority of the remaining proteins were stimulated by 2-4-fold. Greater resolution achieved by pulse-chase labelling and subcellular fractionation showed that, of 32 major proteins localized to insulin secretory granules, the biosynthesis of 25 were stimulated 15-30-fold by glucose. By contrast, only eight of 160 proteins in the soluble fraction showed a response of similar magnitude. It is concluded that there is a major and co-ordinated activation of the biosyntheses of proteins destined for secretory granules, which most likely occurs at the level of translational initiation and signal-recognition-particle-mediated translocation into the endoplasmic reticulum lumen. However, it is clear that not all granule proteins, or the majority of proteins translocated across the endoplasmic reticulum membrane, are affected in an equivalent manner. In addition, the synthesis of a small number of cytosolic proteins may be increased markedly by insulinotropic stimuli.
| S-EPMC1149921 | biostudies-other
Project description:A universal plant response to phosphorus deprivation is the up-regulation of a diverse array of phosphatases. As reported recently, the AtPECP1 gene encodes a phosphatase with in vitro substrate specificity for phosphoethanolamine and phosphocholine. The putative substrates suggested that AtPECP1 is related to phospholipid metabolism; however, the biological function of AtPECP1 is as yet not understood. In addition, whereas lipid remodelling processes as part of the phosphorus starvation response have been extensively studied, knowledge of the polar head group metabolism and its regulation is lacking. We found that AtPECP1 is expressed in the cytosol and exerts by far its strongest activity in roots of phosphate-starved plants. We established a novel LC-MS/MS-based method for the quantitative and simultaneous measurement of the head group metabolites. The analysis of Atpecp1 null mutants and overexpression lines revealed that phosphoethanolamine, but not phosphocholine is the substrate of AtPECP1 in vivo. The impact on head group metabolite levels is greatest in roots of both loss-of-function and gain-of-function transgenic lines, indicating that the biological role of AtPECP1 is mainly restricted to roots. We suggest that phosphoethanolamine hydrolysis by AtPECP1 during Pi starvation is required to down-regulate the energy-consuming biosynthesis of phosphocholine through the methylation pathway.
| S-EPMC5853852 | biostudies-other
Project description:Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a 'moonlighting' role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ, implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria.
| S-EPMC7209980 | biostudies-literature