Rosemary Cornell - Academia.edu (original) (raw)
Papers by Rosemary Cornell
Advances in Lipobiology, 1996
Publisher Summary CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme ... more Publisher Summary CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine (PC) metabolism. It catalyzes the transfer of a cytidyl group from CTP to phosphocholine to form CDPcholine, the head group carrier molecule. CDPcholine is subsequently attacked by diacylglycerol (DAG), releasing CMP and forming PC. This pathway for forming PC is the dominant pathway in all animal cells. PC is the source of DAG production via phospholipase D and phosphatidic acid phosphatase in response to bombesin, epinephrine, vasopressin, cholecystokinin, and other agonists. In these pathways, phosphatidylinositol bisphosphate- (PIP 2 ) specific phospholipase C (PLC) generates the first wave of DAG production, followed by more sustained production of DAG via the hydrolysis of PC. CT may also be involved in attenuation of the DAG signal. The activity of CT is regulated by many factors. The addition to cultured cells of phorbol esters, phospholipases, fatty acids, diacylglycerol, calcium ionophore, and CSF-1 all lead to stimulation of CT activity.
Phosphatidylcholine Metabolism, 2020
Journal of Biological Chemistry, 1989
5 104-5 110). Sodium dodecyl sulfate-poIyacrylamide gel electrophoresis with or without 8-mercapt... more 5 104-5 110). Sodium dodecyl sulfate-poIyacrylamide gel electrophoresis with or without 8-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiolcleavable cross-linking reagents, dithiobis(succimidy1 propionate) or dimethyL3,3'-dithiobispropionirnidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.
Journal of Biological Chemistry, 1990
We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine ac... more We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.
Journal of Biological Chemistry, 1994
Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were express... more Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were expressed in COS cells. The specific activity of CT in the microsomes increased approximately 20- or 100-fold after transfection with CT-1 or CT-2, respectively, but there was only a 3-5 fold increase in the rate of [3H]choline or [3H]glycerol incorporation into phosphatidylcholine (PC). The phosphocholine pool decreased approximately 40% in keeping with a stimulation of the CT-catalyzed reaction. The CDP-choline pool increased 12-fold suggesting that the conversion of CDP-choline to PC, catalyzed by cholinesphosphotransferase, could not keep pace with the CT-catalyzed reaction. This could account for the discrepancy between the increases in the amount of active (membrane-bound) CT and the rate of PC synthesis. Incubation of CT-transfected cells with sodium oleate to increase the supply of cellular diacylglycerol resulted in a further 2-fold increase in the rate of PC synthesis. This suggests that the diacylglycerol supply may be a limiting factor in the degree of stimulation of PC synthesis in CT-transfected COS cells. Despite the increased rate of PC synthesis, the total cellular PC mass increased only 17%, due to a 3-fold acceleration of the PC degradation rate. To determine which degradative pathway for PC was accelerated in the CT-transfected cells, we measured the pool sizes of several catabolites. Neither diacylglycerol nor phosphatidic acid mass was altered. The pool of glycerophosphocholine (GPC) was increased approximately 4-fold, and there was elevated release of GPC from the CT-transfected cells. The turnover of choline in GPC and lyso-PC was very slow compared with that of choline, phosphocholine, or CDP-choline, suggesting that GPC and lyso-PC were derived from slowly degraded choline-labeled PC. The metabolism of GPC and lyso-PC was stimulated in the cells over-expressing CT. These data suggest that PC synthesis and degradation are coordinated and that PC catabolism involving PC-->lyso-PC-->GPC is accelerated in COS cells overexpressing CT.
Journal of Biological Chemistry, 1991
Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the ce... more Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.
Journal of Biological Chemistry, 1985
The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcopl... more The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcoplasmic reticulum was investigated in differentiating L6 cells in culture. The rates of lipid synthesis and turnover in L6 showed no large variations over the course of differentiation from myoblasts to myotubes while the rate of synthesis of the sarcoplasmic reticulum Ca2+-ATPase steadily increased. Removal of choline from the culture medium after the onset of fusion resulted in a 2-fold inhibition of phosphatidylcholine (PC) synthesis and a 40-50% reduction in total cellular PC content within 36 h. The synthesis and content of phosphatidylethanolamine also declined subsequent to the effect on PC. The amount of newly synthesized phospholipid in the microsomal fraction also decreased 50% in choline-deprived cells. Choline deprivation of myotubes for up to 4 days had no effect on the rates of synthesis of the Ca2+-ATPase or two intrinsic glycoproteins of 53,000 and 160,000 daltons. The newly synthesized proteins were incorporated into PC-deficient microsomal membranes. The synthesis of total cellular protein and total membrane protein was not altered, thus phospholipid:protein ratios declined 2-fold. These observations suggest that the assembly of the sarcoplasmic reticulum is not tightly coordinated with the rate of phospholipid synthesis.
Journal of Biological Chemistry, 1994
Limited chymotrypsin proteolysis of CTPwhosphocholine cytidylyltransferase (CT; EC 2.7.7.15) prod... more Limited chymotrypsin proteolysis of CTPwhosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time c o m e of chymotrypsin proteolysis showed a progression in digestion as follows: 42 + 39-* 35 + 30 + 28 + 26 m a. The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated w i t h the presence of a putative amphipathic a-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosp~tidylcholin~sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to c h y m o t~s i n degradation increased when CT was bound to the strongly activating lipid vesicles phoEphatidylcho~~ oleic acid (1:l) and phoaphatidylcholin~phosphatidylglycerol (1:l). Conversion of the 39-and 36-kDa fragmenb, which contain the intact helix-1, to the 30-, 2&, and 26-ma bands, which lack helix-1, required longer proteolysis times, suggesting that this helical domain is more shielded from solvent upon membrane binding. These results support the theory that CT has a bipartite tertiary structure composed of a globular Nterminal domain and an extended C-terminal domain and that CT interacts with membranes via its putative amphipathic helix which intercalates into the membrane bilayer of activating phospholipids. CTPphosphocholine cytidylyltransferase (CT EC 2.7.7.15)l is a key regulatory enzyme in the biosynthetic pathway of phosphatidylcholine in higher eukaryotes. It exists in an inactive soluble form and an active membrane-bound form. Both forms appear to be homodimers of a 42-kDa monomer (1, 2). Two major types of regulation have been identified which affect the * This work was supported by a grant from the Natural Science and this article were defrayed in part by the payment of page charges. This Engineering Research Council of Canada. The costs of publication of with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked ~~v e~~e~~~n in accordance $ Recipient of a Simon Frawr University Graduate Research Fellowship and MacM~l~-Bloedel Graduate Scholarships.
Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive dis... more Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibr...
Developmental Cell, 2018
Enzyme control by their products facilitates cellular homeostasis, but for phospholipids, feedbac... more Enzyme control by their products facilitates cellular homeostasis, but for phospholipids, feedback mechanisms also arise from changes in membrane physical properties. In this issue of Developmental Cell, Haider et al. (2018) show that in many actively growing cells, an enzyme of phosphatidylcholine synthesis senses lipid packing in the nuclear membrane.
Journal of Biological Chemistry, 2018
CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine... more CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα (PCYT1A) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could ...
Biochemical Journal, 1995
The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored b... more The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II,...
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acylt... more The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes were undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was &fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987
Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulati... more Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP : phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, SO-W% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyhransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCI. These results suggest that a variety of compounds with differing hea dgroup size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.
Biochemical Society Transactions, 1998
Biochemical Journal, 1992
The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were ov... more The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 micro...
European Journal of Biochemistry, 1998
The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cyti... more The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10743-10750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP-binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100-fold lower than that of wild-type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition-state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the tau nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition-state stabilization.
Trends in Biochemical Sciences
ABSTRACT
Advances in Lipobiology, 1996
Publisher Summary CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme ... more Publisher Summary CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine (PC) metabolism. It catalyzes the transfer of a cytidyl group from CTP to phosphocholine to form CDPcholine, the head group carrier molecule. CDPcholine is subsequently attacked by diacylglycerol (DAG), releasing CMP and forming PC. This pathway for forming PC is the dominant pathway in all animal cells. PC is the source of DAG production via phospholipase D and phosphatidic acid phosphatase in response to bombesin, epinephrine, vasopressin, cholecystokinin, and other agonists. In these pathways, phosphatidylinositol bisphosphate- (PIP 2 ) specific phospholipase C (PLC) generates the first wave of DAG production, followed by more sustained production of DAG via the hydrolysis of PC. CT may also be involved in attenuation of the DAG signal. The activity of CT is regulated by many factors. The addition to cultured cells of phorbol esters, phospholipases, fatty acids, diacylglycerol, calcium ionophore, and CSF-1 all lead to stimulation of CT activity.
Phosphatidylcholine Metabolism, 2020
Journal of Biological Chemistry, 1989
5 104-5 110). Sodium dodecyl sulfate-poIyacrylamide gel electrophoresis with or without 8-mercapt... more 5 104-5 110). Sodium dodecyl sulfate-poIyacrylamide gel electrophoresis with or without 8-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiolcleavable cross-linking reagents, dithiobis(succimidy1 propionate) or dimethyL3,3'-dithiobispropionirnidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.
Journal of Biological Chemistry, 1990
We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine ac... more We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.
Journal of Biological Chemistry, 1994
Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were express... more Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were expressed in COS cells. The specific activity of CT in the microsomes increased approximately 20- or 100-fold after transfection with CT-1 or CT-2, respectively, but there was only a 3-5 fold increase in the rate of [3H]choline or [3H]glycerol incorporation into phosphatidylcholine (PC). The phosphocholine pool decreased approximately 40% in keeping with a stimulation of the CT-catalyzed reaction. The CDP-choline pool increased 12-fold suggesting that the conversion of CDP-choline to PC, catalyzed by cholinesphosphotransferase, could not keep pace with the CT-catalyzed reaction. This could account for the discrepancy between the increases in the amount of active (membrane-bound) CT and the rate of PC synthesis. Incubation of CT-transfected cells with sodium oleate to increase the supply of cellular diacylglycerol resulted in a further 2-fold increase in the rate of PC synthesis. This suggests that the diacylglycerol supply may be a limiting factor in the degree of stimulation of PC synthesis in CT-transfected COS cells. Despite the increased rate of PC synthesis, the total cellular PC mass increased only 17%, due to a 3-fold acceleration of the PC degradation rate. To determine which degradative pathway for PC was accelerated in the CT-transfected cells, we measured the pool sizes of several catabolites. Neither diacylglycerol nor phosphatidic acid mass was altered. The pool of glycerophosphocholine (GPC) was increased approximately 4-fold, and there was elevated release of GPC from the CT-transfected cells. The turnover of choline in GPC and lyso-PC was very slow compared with that of choline, phosphocholine, or CDP-choline, suggesting that GPC and lyso-PC were derived from slowly degraded choline-labeled PC. The metabolism of GPC and lyso-PC was stimulated in the cells over-expressing CT. These data suggest that PC synthesis and degradation are coordinated and that PC catabolism involving PC-->lyso-PC-->GPC is accelerated in COS cells overexpressing CT.
Journal of Biological Chemistry, 1991
Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the ce... more Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.
Journal of Biological Chemistry, 1985
The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcopl... more The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcoplasmic reticulum was investigated in differentiating L6 cells in culture. The rates of lipid synthesis and turnover in L6 showed no large variations over the course of differentiation from myoblasts to myotubes while the rate of synthesis of the sarcoplasmic reticulum Ca2+-ATPase steadily increased. Removal of choline from the culture medium after the onset of fusion resulted in a 2-fold inhibition of phosphatidylcholine (PC) synthesis and a 40-50% reduction in total cellular PC content within 36 h. The synthesis and content of phosphatidylethanolamine also declined subsequent to the effect on PC. The amount of newly synthesized phospholipid in the microsomal fraction also decreased 50% in choline-deprived cells. Choline deprivation of myotubes for up to 4 days had no effect on the rates of synthesis of the Ca2+-ATPase or two intrinsic glycoproteins of 53,000 and 160,000 daltons. The newly synthesized proteins were incorporated into PC-deficient microsomal membranes. The synthesis of total cellular protein and total membrane protein was not altered, thus phospholipid:protein ratios declined 2-fold. These observations suggest that the assembly of the sarcoplasmic reticulum is not tightly coordinated with the rate of phospholipid synthesis.
Journal of Biological Chemistry, 1994
Limited chymotrypsin proteolysis of CTPwhosphocholine cytidylyltransferase (CT; EC 2.7.7.15) prod... more Limited chymotrypsin proteolysis of CTPwhosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time c o m e of chymotrypsin proteolysis showed a progression in digestion as follows: 42 + 39-* 35 + 30 + 28 + 26 m a. The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated w i t h the presence of a putative amphipathic a-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosp~tidylcholin~sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to c h y m o t~s i n degradation increased when CT was bound to the strongly activating lipid vesicles phoEphatidylcho~~ oleic acid (1:l) and phoaphatidylcholin~phosphatidylglycerol (1:l). Conversion of the 39-and 36-kDa fragmenb, which contain the intact helix-1, to the 30-, 2&, and 26-ma bands, which lack helix-1, required longer proteolysis times, suggesting that this helical domain is more shielded from solvent upon membrane binding. These results support the theory that CT has a bipartite tertiary structure composed of a globular Nterminal domain and an extended C-terminal domain and that CT interacts with membranes via its putative amphipathic helix which intercalates into the membrane bilayer of activating phospholipids. CTPphosphocholine cytidylyltransferase (CT EC 2.7.7.15)l is a key regulatory enzyme in the biosynthetic pathway of phosphatidylcholine in higher eukaryotes. It exists in an inactive soluble form and an active membrane-bound form. Both forms appear to be homodimers of a 42-kDa monomer (1, 2). Two major types of regulation have been identified which affect the * This work was supported by a grant from the Natural Science and this article were defrayed in part by the payment of page charges. This Engineering Research Council of Canada. The costs of publication of with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked ~~v e~~e~~~n in accordance $ Recipient of a Simon Frawr University Graduate Research Fellowship and MacM~l~-Bloedel Graduate Scholarships.
Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive dis... more Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibr...
Developmental Cell, 2018
Enzyme control by their products facilitates cellular homeostasis, but for phospholipids, feedbac... more Enzyme control by their products facilitates cellular homeostasis, but for phospholipids, feedback mechanisms also arise from changes in membrane physical properties. In this issue of Developmental Cell, Haider et al. (2018) show that in many actively growing cells, an enzyme of phosphatidylcholine synthesis senses lipid packing in the nuclear membrane.
Journal of Biological Chemistry, 2018
CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine... more CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα (PCYT1A) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could ...
Biochemical Journal, 1995
The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored b... more The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II,...
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acylt... more The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes were undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was &fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987
Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulati... more Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP : phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, SO-W% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyhransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCI. These results suggest that a variety of compounds with differing hea dgroup size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.
Biochemical Society Transactions, 1998
Biochemical Journal, 1992
The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were ov... more The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 micro...
European Journal of Biochemistry, 1998
The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cyti... more The HXGH motif of CTP:phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10743-10750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP-binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100-fold lower than that of wild-type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition-state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the tau nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition-state stabilization.
Trends in Biochemical Sciences
ABSTRACT