RESUMO
CTP:Phosphocholine cytidylyltransferase (CT) catalyzes the key step in phosphatidylcholine (PC) synthesis. CT is activated by binding to certain lipid membranes. The membrane binding affinity of CT can vary from micromolar to millimolar K(d), depending on the lipid composition of the target membrane. Class II CT activators like diacylglycerols and unsaturated phosphatidylethanolamines (PE) favor inverted lipid phase formation. The mechanism(s) governing CT's association with class II lipid membranes and subsequent activation are relatively unknown. We measured CT activation by vesicles composed of PC and one of three unsaturated PEs, dioleoylglycerol (DOG), or cholesterol. For each lipid system, we estimated the stored curvature strain energy of the monolayer when confined to a relatively flat bilayer. CT binding and activation correlate very well with the curvature strain energy of several chemically distinct class II lipid systems, with the exception of those containing cholesterol, in which CT activation was less than the increase in curvature strain. CT activation by membranes containing DOG was reversed by inclusion of specific lysolipids, which reduce curvature strain energy. LysoPC, which has a larger positive curvature than lysoPE, produced greater inhibition of CT activation. Stored curvature strain energy is thus an important determinant of CT activation. Membrane interfacial polarity was investigated using a membrane-anchored fluorescent probe. Decreases in quenching of this interfacial probe by doxyl-PCs in class II membranes suggest the probe adopts a more superficial membrane location. This may reflect an increased surface hydrophobicity of class II lipid membranes, implying a role for surface dehydration in CT's interactions with membranes containing class II lipids. Cholesterol, a poor activator of CT, did not affect the positioning of the polarity-sensitive probe, suggesting that one reason for its ineffectiveness is an inability to enhance surface hydrophobicity.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Lipídeos de Membrana/metabolismo , Cumarínicos , Ativação Enzimática , Corantes Fluorescentes , Lipossomos , Lisofosfolipídeos/metabolismo , Lipídeos de Membrana/química , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Phosphatidylcholine (PC) synthesis in animal cells is generally controlled by cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase (CCT). This enzyme is amphitropic, that is, it can interconvert between a soluble inactive form and a membrane-bound active form. The membrane-binding domain of CCT is a long amphipathic alpha helix that responds to changes in the physical properties of PC-deficient membranes. Binding of this domain to membranes activates CCT by relieving an inhibitory constraint in the catalytic domain. This leads to stimulation of PC synthesis and maintenance of membrane PC content. Surprisingly, the major isoform, CCT alpha, is localized in the nucleus of many cells. Recently, a new level of its regulation has emerged with the discovery that signals that stimulate PC synthesis recruit CCT alpha from an inactive nuclear reservoir to a functional site on the endoplasmic reticulum.
Assuntos
Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Ativação Enzimática , Animais , Domínio Catalítico , Linhagem Celular , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Metabolismo dos Lipídeos , Modelos Biológicos , Modelos Moleculares , Fosfatidilcolinas/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
The enzyme CTP:phosphocholine cytidylyltransferase (CT) binds reversibly to membranes and is active only in its membrane-bound form. Membrane lipid composition influences the equilibrium between its soluble and membrane-bound forms. Whereas the enzyme is not activated by phosphatidylcholine (PC) vesicles, it is activated by PC vesicles that have been oxidized with HClO(4) [Drobnies, A. E., et al. (1998) Biochim. Biophys. Acta 1393, 90-98]. Here we explore the mechanism of activation of CT by a PC oxidized with lipoxidase. Multilamellar vesicles (MLVs) containing > or =5 mol % oxidized 1-palmitoyl-2-arachidonoylPC (PAPC) progressively activated the enzyme, which was fully activated by 25 mol % oxidized PC. The effect of oxidized PAPC on lipid order was investigated by (2)H NMR, using MLVs containing PAPC perdeuterated on the palmitoyl chain. Spectral depaking generated order parameter profiles along the sn-1 chain. The average order parameter (S(CD)) in the plateau region at 37 degrees C decreased from 0.18 to 0.15 with increasing percent of oxidized PAPC (0-25%). The change in S(CD) was even greater near the end of the palmitoyl chain. CT activation was inversely related to lipid order. The major component of the lipoxidase-oxidized PAPC was purified and characterized by mass spectrometry and NMR. This component, 1-palmitoyl-2-(11,15-dihydroxy)eicosatrienoylPC (dihydroxyPAPC), incorporated into PAPC MLVs, also stimulated CT activity and reduced the lipid order parameter. Both effects were reversed by egg sphingomyelin. We propose that CT activation by oxidized PAPC is mediated by effects on lipid packing perturbations. This is the first study to report the effects of a purified oxidized PC on the orientational order along the acyl chain and to correlate the lipid disordering of the oxidized PC with the activation of a membrane-associated regulatory enzyme.
Assuntos
Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Catálise , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Deutério/química , Deutério/metabolismo , Ativação Enzimática/efeitos dos fármacos , Lipoproteínas LDL/química , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas LDL/metabolismo , Lipoxigenase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Éteres Fosfolipídicos/química , Éteres Fosfolipídicos/isolamento & purificação , Éteres Fosfolipídicos/metabolismo , Glycine max/enzimologia , Esfingomielinas/químicaRESUMO
The transition from quiescence (G(0)) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at approximately 10 min, peaking at approximately 3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated approximately 3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until approximately 4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G(0) --> G(1) transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle.
Assuntos
Núcleo Celular/enzimologia , Colina-Fosfato Citidililtransferase/metabolismo , Retículo Endoplasmático/enzimologia , Fase G1 , Fosfatidilcolinas/biossíntese , Fase de Repouso do Ciclo Celular , Transporte Biológico , Linhagem Celular , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Fosfatidilcolinas/metabolismo , FosforilaçãoRESUMO
What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding.
Assuntos
Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Conformação Proteica , Transdução de SinaisRESUMO
CT responds to properties of PC-depleted membranes: increased negative charge density, which concentrates the enzyme at the membrane surface, and lipid packing perturbations, which create holes in the membrane surface into which the hydrophobic side chains of the amphipathic helix of domain M can intercalate. The PC-deficient lipid surface appears capable of catalysing the folding of domain M into an alpha-helix. The determinants on domain M which create a preference for anionic lipids are: (i) strips of interfacial lysines; (ii) three serines within the non-polar face; (iii) three interfacial glutamates whose protonation state appears to be sensitive to the surface charge. Phosphorylation of the domain adjacent to domain M decreases the membrane affinity of the amphipathic helix, perhaps by an ion-pairing competition. The mechanism whereby the stabilization of an alpha-helical conformation of domain M is transduced into a conformational change in the catalytic domain is the key question for future exploration.
Assuntos
Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipídeos/químicaRESUMO
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.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Citidina Trifosfato/metabolismo , Histidina , Animais , Sítios de Ligação , Células COS , Colina-Fosfato Citidililtransferase/genética , Estabilidade Enzimática , Ligação de Hidrogênio , Cinética , Fígado/enzimologia , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Desnaturação Proteica , Ratos , TransfecçãoRESUMO
CTP:phosphocholine cytidylyltransferase (CT) catalyzes a rate-limiting, regulatory step in mammalian biosynthesis of phosphocholine (PC). Anionic phospholipids, fatty acids and diacylglycerol activate CT and promote its intercalation into the lipid bilayer, whereas zwitterionic phospholipids such as phosphatidylcholines do not. We investigated the effectiveness of polyunsaturated phosphatidylcholines as CT activators after hypochlorite oxidation. Detection and quantitation of oxidized PCs were evaluated by thin layer chromatography, high performance liquid chromatography, and conjugated dienes. Purified CT was assayed in the presence of multilamellar vesicles, containing variable concentrations of oxidized and parent PCs. The results demonstrate that particular species of oxidized PCs activate CT as potently as anionic lipids. The greater the number of double bonds available for oxidation in the fatty acid at the sn-2 position of the PC, the more effective was the oxidized PC as an activator of CT. Oxidized phospholipids at 1:1 bleach/lipid activated CT in the following order: PAPC>PL3PC>PL2PC compared to unoxidized controls. Since oxidized phospholipids decrease bilayer order (M.L. Wratten et al., Biochemistry 31 (1992) 10901-10907) these results are consistent with the activation of CT by perturbations of lipid bilayer packing.
Assuntos
Ácido Hipocloroso/farmacologia , Nucleotidiltransferases/metabolismo , Fosfatidilcolinas/farmacologia , Ativação Enzimática , Nucleotidiltransferases/isolamento & purificação , OxirreduçãoRESUMO
We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Colina-Fosfato Citidililtransferase/química , Dicroísmo Circular , Lipossomos/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Pressão , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Propriedades de SuperfícieRESUMO
The regulation of membrane binding and activity of purified CDP:phosphocholine cytidylyl-transferase (CT) by lipid activators and enzyme dephosphorylation was examined. The binding of CT to membranes was analyzed using sucrose-loaded vesicles (SLVs). Binding to phosphatidylcholine vesicles was not detected even at a lipid:protein ratio of approximately 2000 (1 mM PC). CT bound to vesicles containing anionic lipids with apparent molar partition coefficients between 2 x 10(5) and 2 x 10(6), depending on the vesicle charge. The vesicle binding and activation of CT showed very similar sigmoidal dependencies on the lipid negative charge. In addition, diacylglycerol interacted synergistically with anionic phospholipids to stimulate both binding and activation at lower mole percent anionic lipid. These results demonstrate parallel requirements for binding and activity. Dephosphorylation of CT without destabilization was accomplished using the catalytic subunit of protein phosphatase 1. Dephosphorylated CT required a lower mole percent anionic phospholipid than phosphorylated CT for binding to and activation by SLVs. The combination of 10 mol % diacylglycerol and enzyme dephosphorylation shifted the mole percent phosphatidic acid required for half-maximal activation from 25% to 12%. These results suggest a mechanism whereby large changes in CT activity can result from changes in the phosphorylation state combined with small alterations in the membrane content of diacylglycerol. We propose a mechanism whereby dephosphorylation on the domain adjacent to the membrane binding domain increases the affinity of the latter for a negatively charged membrane surface.
Assuntos
Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Colina-Fosfato Citidililtransferase , Diglicerídeos/farmacologia , Sinergismo Farmacológico , Ativação Enzimática , Membranas/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosforilação , Ligação ProteicaRESUMO
CTP:phosphocholine cytidylyltransferase (CT), the rate controlling enzyme in phosphatidylcholine biosynthesis, is activated by reversible membrane binding. To investigate the membrane binding mechanism of CT, we have used the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[l25I]iodophenyl)diazirine ([125I]TID). Association of CT with phosphatidylcholine/oleic acid (1:1) vesicles was first demonstrated by gel filtration analysis. Upon irradiation, CT was covalently labeled by [125I]TID presented in phosphatidylcholine/oleic acid vesicles. This demonstrates an intercalation of part of the protein into the hydrophobic core of the membrane. To identify the membrane-embedded domain, the chymotrypsin digestion products of [125I]TID labeled CT were analysed. Chymotrypsin digestion produced a set of previously defined N-terminal fragments (Craig, L., Johnson, J.E. and Cornell, R.B. (1994) J. Biol. Chem. 269, 3311), as well as several small C-terminal fragments which react with an anti-peptide antibody raised against the proposed amphipathic alpha-helix. All fragments containing the amphipathic helical region of the enzyme had [125I]TID label associated, while the chymotryptic fragment which lacked this region was not highly labeled. Similar fragment labeling patterns were produced when [125I]TID was presented in phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol vesicles, suggesting that the same domain of CT mediates binding to membranes containing either of the two lipid activators. A 62-residue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with [125I]TID, demonstrating its ability to intercalate independently of the rest of the protein. These results indicate a membrane-binding mechanism for cytidylyltransferase involving the intercalation of the amphipathic alpha-helix region into the hydrophobic acyl chain core of the activating membrane.
Assuntos
Azirinas , Membrana Celular/enzimologia , Reagentes de Ligações Cruzadas , Nucleotidiltransferases/química , Animais , Colina-Fosfato Citidililtransferase , Quimotripsina , Diglicerídeos/metabolismo , Lipídeos de Membrana/metabolismo , Peso Molecular , Ácido Oleico/metabolismo , Fragmentos de Peptídeos/química , Peptídeos/síntese química , Fosfatidilcolinas/metabolismo , Fotoquímica , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes , Análise de SequênciaRESUMO
It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic alpha-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly alpha-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined alpha-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an alpha-helix from Glu243 to Lys266, a well-structured bend of about 50 degrees at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces.
Assuntos
Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/enzimologia , Colina-Fosfato Citidililtransferase , Dicroísmo Circular , Gráficos por Computador , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , RatosRESUMO
The effect of mutations in the proposed catalytic domain of CTP:phosphocholine cytidylyltransferase was investigated by constructing the single mutants CT-S91 and CT-C114 from the double mutant CT-S91C114, previously shown to have 4-fold lower than wild-type activity [Walkey, C.R., Kalmar, G. B., & Cornell, R. B. (1994) J. Biol. Chem. 269, 5742-5749]. The constructs were overexpressed in COS cells. The mutation Gly-91 to Ser-91 was found to be responsible for the decreased activity, whereas Ser-114 to Cys-114 had no effect. An alanine substitution at position 91, CT-A91, had a lesser effect on cytidylyltransferase activity. CT-S91 and CT-WT were purified from COS cells, and their kinetic constants were determined. CT-S91 had a 4-fold lower Vmax, and a K(m) for CTP 25-fold higher than the wild-type enzyme, suggesting that substitution of Gly-91 with serine interferes with CTP binding. The K(m) for phosphocholine was not affected in the CT-S91 mutant. There was no difference in the chymotrypsin sensitivities of CT-S91 and CT-WT, indicating that the mutation did not cause a global change in protein structure. However, the CT-S91 activity was more susceptible to inhibition by the denaturant urea than that of CT-WT, indicative of a perturbation of the active site folding. Gly-91 resides in the local sequence HSGH, which has been proposed to be a CTP-binding motif in the novel cytidylyltransferase superfamily [Bork, P., Holm, L., Koonin, E.V., & Sander, C. (1995) Proteins: Struct., Funct., Genet. 22, 259-266]. Our results represent the first experimental validation of this hypothesis.
Assuntos
Glicina/química , Nucleotidiltransferases/metabolismo , Serina/química , Animais , Sequência de Bases , Linhagem Celular , Colina-Fosfato Citidililtransferase , Quimotripsina/metabolismo , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Desnaturação Proteica , Especificidade por Substrato , Ureia/químicaRESUMO
The contributions of electrostatic and hydrophobic interactions in the activation of cytidylyl-transferase (CT) by various negatively charged lipids were analyzed using small unilamellar or multilamellar vesicles (SUVs or MLVs). The activation of CT by SUVs containing increasing mole percentages of anionic phospholipids varied in proportion to the net charge associated with the polar head group, suggesting an electrostatic component to the activation. However, increasing ionic strength to neutralize the surface charge enhanced the potency of SUVs containing PA or PG, suggesting that the hydrophobic effect is a stronger force than electrostatics in driving the interaction of CT with SUVs. On the other hand, electrostatics played a more important role in the activation by MLVs. Increasing ionic strength decreased the potency of MLVs containing PG. CT bound to MLVs in the gel state, but was inactive; the enzyme was only active when the MLVs were in the liquid-crystalline state, suggesting an intercalation event. Lowering the pH from 7.4 to 6.2 resulted in a decrease in the negative surface charge required for activation. The binding of CT to PG vesicles was enhanced at acidic pH. The results suggest that at pH 6.2 one or more amino acids on CT that are involved in lipid binding would be protonated. This could enhance the electrostatic effect by increasing the positive charge on CT, or it could enhance the hydrophobic effect by decreasing the negative charge on CT. In addition, maximal activity of CT was decreased at the lower pH, suggesting that active site residues may also be affected. CT was activated by the synergistic interaction of diacylglycerol and anionic phospholipid in SUVs. The synergy between DG and PA at low concentrations suggests the possibility that these second messenger lipids could concertedly regulate CT and thus PC synthesis in response to agonists that stimulate PC hydrolysis via phospholipases C and/or D.
Assuntos
Diglicerídeos/farmacologia , Lipossomos , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Fosfolipídeos/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Baculoviridae , Colina-Fosfato Citidililtransferase , Eletroquímica , Ativação Enzimática , Concentração de Íons de Hidrogênio , Insetos , Cinética , Nucleotidiltransferases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sacarose , TransfecçãoRESUMO
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, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.
Assuntos
Fígado/enzimologia , Nucleotidiltransferases/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caseína Quinase II , Divisão Celular , Chlorocebus aethiops , Colina-Fosfato Citidililtransferase , Primers do DNA , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Isoenzimas/metabolismo , Rim , Cinética , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/análise , Nucleotidiltransferases/biossíntese , Fosfopeptídeos/análise , Fosforilação , Mutação Puntual , Reação em Cadeia da Polimerase , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Human CTP:phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library. Initially two degenerate oligonucleotide primers derived from the sequence of the rat liver CT cDNA were used to amplify a centrally located 230 bp fragment. Subsequently overlapping 5' and 3' fragments were amplified, each using one human CT primer and one vector-specific primer. Two cDNAs encoding the entire translated domain were also amplified. The human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary). The region which deviates most from the rat liver CT sequence is near the C-terminus, where 7 changes are clustered within 34 residues (345-359), of the putative phosphorylation domain. The region of the proposed catalytic domain (residues 75-235) is 100% identical with the rat liver sequence. Significant homology was observed between the proposed catalytic domain of CT and the Saccharomyces cerevisiae MUQ1 gene product, and between the proposed amphipathic alpha-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein. There are several shared characteristics of these amphipathic helices. An approx. 42,000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones. The specific activity of the HCT in COS cell homogenates was the same as that of analogously expressed rat liver CT. The activity of HCT was lipid dependent. The soluble form was activated 3 to 4-fold by anionic phospholipids and by oleic acid or diacylglycerol-containing PC vesicles.
Assuntos
Nucleotidiltransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Colina-Fosfato Citidililtransferase , Cricetinae , Cricetulus , Primers do DNA/química , Ativação Enzimática , Humanos , Metabolismo dos Lipídeos , Camundongos , Dados de Sequência Molecular , Fosforilação , Estrutura Secundária de Proteína , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
We conducted a prospective study to evaluate early complications and complaints of 60 patients who underwent laparoscopic transperitoneal hernia repair at our institution. Average follow-up was 9 months. Patients graded levels and duration of postoperative pain subjectively. Nine patients (15%) had complications of anterior/medial thigh numbness, 4 (6.7%) scrotal swelling, 4 (6.7%) scrotal ecchymosis, 3 (5%) hematoma, 2 (3.3%) prolonged sensation of tightness/pressure, 1 (1.7%) seroma, 1 (1.7%) urinary retention, and 1 (1.7%) pain with intercourse. Twenty-six (43%) had no postoperative complaints. Overall, 57 (95%) stated they were satisfied with their repair and would recommend the laparoscopic technique. Fifty-five patients (92%) returned to basic activities of daily living in less than 2 weeks. Thirty-five (73%) of the 48 patients who were employed returned to work within 3 weeks. In comparison, only 7 (29%) of 24 patients in an open hernia repair group resumed normal activity during the first 2 postoperative weeks, and only 3 (14%) of the 21 employed patients in this group returned to work at 3 weeks. The laparoscopic and traditional open herniorrhaphy methods were compared in terms of operating room time and cost. The average unilateral laparoscopic repair (n = 51) cost $3,094 and lasted 81 minutes. Bilateral laparoscopy procedures (n = 9) averaged $3,774 and 110 minutes. Unilateral traditional hernia repairs (n = 24) had an average cost of $1,990 and duration of 69 minutes. In follow-up ranging from 2 to 28 months, there has been only 1 recurrence to report in the laparoscopic group (1.7%). All patients continue to be followed to determine long-term recurrence risks.
Assuntos
Hérnia Inguinal/cirurgia , Laparoscopia/efeitos adversos , Laparoscopia/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peritônio , Estudos Prospectivos , Resultado do TratamentoRESUMO
A peptide corresponding to a portion of the amphipathic alpha-helical region of CTP:phosphocholine cytidylyltransferase was synthesized. This region of the enzyme was proposed to be the membrane-binding domain [Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R., & Cornell, R.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6029]. We have shown that the peptide is physically associated with PG vesicles. CD of the peptide in buffer suggested a primarily random structure, while, in the presence of trifluoroethanol, the peptide was alpha-helical. Anionic lipid vesicles promoted an alpha-helical conformation, whereas neutral or cationic lipid vesicles did not alter the random structure of the peptide, suggesting a selective stabilization of the alpha-helix by anionic membranes. The fluorescence of the single tryptophan residue, which lies on the hydrophobic face of the amphipathic alpha-helix, was studied. Anionic lipid vesicles specifically induced a shift in the fluorescence to a lower wavelength. Fluorescence quenching by the aqueous-phase quencher, I-, and the lipid-phase quencher 9,10-dibromo-PC was used to determine the accessibility of the tryptophan to each of these environments. The presence of anionic lipid vesicles, but not nonanionic lipid vesicles, decreased the quenching by I- suggesting that, in the presence of anionic lipids, the tryptophan residue is poorly accessible to the aqueous I-. Dibromo-PC significantly quenched the fluorescence only when present in anionic vesicles, confirming the membrane location of the tryptophan residue and the lipid specificity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Membrana/química , Nucleotidiltransferases/química , Fragmentos de Peptídeos/síntese química , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Ânions , Sítios de Ligação , Bromo , Membrana Celular/enzimologia , Colina-Fosfato Citidililtransferase , Dicroísmo Circular , Iodetos , Proteínas de Membrana/síntese química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
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.
Assuntos
Fígado/enzimologia , Nucleotidiltransferases/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Linhagem Celular , Membrana Celular/enzimologia , Colina/metabolismo , Colina-Fosfato Citidililtransferase , Citosol/enzimologia , Glicerol/metabolismo , Nucleotidiltransferases/genética , Ácido Oleico , Ácidos Oleicos/farmacologia , Ratos , TransfecçãoRESUMO
Limited chymotrypsin proteolysis of CTP:phosphocholine 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 course of chymotrypsin proteolysis showed a progression in digestion as follows: 42-->39-->35-->30-->28-->26 kDa. 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 with the presence of a putative amphipathic alpha-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosphatidylcholine/sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to chymotrypsin degradation increased when CT was bound to the strongly activating lipid vesicles phosphatidylcholine/oleic acid (1:1) and phosphatidylcholine/phosphatidylglycerol (1:1). Conversion of the 39- and 35-kDa fragments, which contain the intact helix-1, to the 30-, 28-, and 26-kDa 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 N-terminal 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.