Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Plant Sci ; 302: 110698, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33288011

RESUMEN

Phosphatidylcholine is a major phospholipid which is shown to be involved in stress adaptation. Phosphatidylcholine increased during dehydration in Craterostigma plantagineum, and therefore we characterized CTP:phosphocholine cytidylyltransferase (CpCCT1), a key regulatory enzyme for phosphatidylcholine synthesis in plants. The CpCCT1 gene from the resurrection plant C. plantagineum was cloned and the amino acid sequence was compared with homologs from other species including yeast and rat. CCT proteins have conserved catalytic and membrane-binding domains while the N-terminal and C-terminal domains have diverged. The tissue specific expression analysis indicated that CpCCT1 is expressed in all tested tissues and it is induced by dehydration and in response to 0.5 M NaCl solutions. In plants exposed to low temperature in the dark, the CpCCT1 transcript increased after 4 h at 4 °C. CpCCT1 expression also increased during mannitol and sorbitol treatments in a concentration dependent manner. Phytohormones such as abscisic acid and indole-3-acetic acid also trigged transcript accumulation. Comparisons of transcript and protein accumulations for different treatments (except for dehydration) suggest transcriptional and translational control mechanisms. Analysis of promoter activity and polysome occupancy suggest that CpCCT1 gene expression is mainly under translational regulation during dehydration.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/metabolismo , Craterostigma/enzimología , Proteínas de Plantas/metabolismo , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/fisiología , Clonación Molecular , Craterostigma/genética , Deshidratación , Regulación de la Expresión Génica de las Plantas , Fosfatidilcolinas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Alineación de Secuencia
2.
Life Sci Alliance ; 3(8)2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32461215

RESUMEN

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40-50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/metabolismo , Gotas Lipídicas/metabolismo , Fosfatidato Fosfatasa/metabolismo , Animales , Células CHO , Núcleo Celular/metabolismo , Citidililtransferasa de Colina-Fosfato/fisiología , Cricetulus , Ácidos Grasos/metabolismo , Humanos , Gotas Lipídicas/fisiología , Membrana Nuclear/metabolismo , Ácido Oléico/metabolismo , Fosfatidato Fosfatasa/fisiología , Fosfatidilcolinas/química , Proteína de la Leucemia Promielocítica/metabolismo , Proteína de la Leucemia Promielocítica/fisiología
3.
Cancer Res ; 76(19): 5634-5646, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27457520

RESUMEN

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634-46. ©2016 AACR.


Asunto(s)
Neoplasias de la Mama/etiología , Colina/metabolismo , Receptor alfa de Estrógeno/fisiología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Citidililtransferasa de Colina-Fosfato/fisiología , Diacilglicerol Colinafosfotransferasa/fisiología , Resistencia a Antineoplásicos , Femenino , Humanos , Células MCF-7 , Metástasis de la Neoplasia , Tamoxifeno/uso terapéutico , Pez Cebra
4.
Biochim Biophys Acta ; 1861(8 Pt B): 847-861, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26747646

RESUMEN

The amphipathic helical (AH) membrane binding motif is recognized as a major device for lipid compositional sensing. We explore the function and mechanism of sensing by the lipid biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CCT). As the regulatory enzyme in phosphatidylcholine (PC) synthesis, CCT contributes to membrane PC homeostasis. CCT directly binds and inserts into the surface of bilayers that are deficient in PC and therefore enriched in lipids that enhance surface charge and/or create lipid packing voids. These two membrane physical properties induce the folding of the CCT M domain into a ≥60 residue AH. Membrane binding activates catalysis by a mechanism that has been partially deciphered. We review the evidence for CCT compositional sensing, and the membrane and protein determinants for lipid selective membrane-interactions. We consider the factors that promote the binding of CCT isoforms to the membranes of the ER, nuclear envelope, or lipid droplets, but exclude CCT from other organelles and the plasma membrane. The CCT sensing mechanism is compared with several other proteins that use an AH motif for membrane compositional sensing. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/química , Citidililtransferasa de Colina-Fosfato/fisiología , Mecanotransducción Celular/fisiología , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína
5.
Prog Lipid Res ; 59: 147-71, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26165797

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-limiting and regulated step in the CDP-choline pathway for the synthesis of phosphatidylcholine (PC) and PC-derived lipids. Control of CCT activity is multi-layered, and includes direct regulation by reversible membrane binding involving a built-in lipid compositional sensor. Thus CCT contributes to phospholipid compositional homeostasis. CCT also modifies the curvature of its target membrane. Knowledge of CCT structure and regulation of its catalytic function are relatively advanced compared to many lipid metabolic enzymes, and are reviewed in detail. Recently the genetic origins of two human developmental and lipogenesis disorders have been traced to mutations in the gene for CCTα.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Fosfatidilcolinas/biosíntesis , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Secuencia Conservada , Humanos , Gotas Lipídicas/metabolismo , Lipoproteínas/fisiología , Datos de Secuencia Molecular , Neurogénesis , Procesamiento Proteico-Postraduccional , Transporte de Proteínas
6.
Hypertension ; 65(2): 430-9, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25452470

RESUMEN

C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation before symptoms in patients with preeclampsia, a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in preeclampsia are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals when compared with nulligravid women and is further increased in patients with preeclampsia. These findings led us to discover further that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in patients with preeclampsia. Next, we demonstrated that injection of CRP induces preeclampsia features, including hypertension (157 mm Hg CRP treated versus 119 mm Hg control), proteinuria (35.0 mg/µg CRP treated versus 14.1 mg/µg control), kidney, and placental damage and increased levels of sFlt-1 in pregnant mice but not in nonpregnant mice. Our study implicates that phosphocholine transferase, a placental-specific enzyme post-translationally modifying neurokinin B, is essential for the pathogenic role of CRP in preeclampsia through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight on the pathogenic role of CRP in preeclampsia and highlighted innovative therapeutic strategies.


Asunto(s)
Proteína C-Reactiva/fisiología , Citidililtransferasa de Colina-Fosfato/fisiología , Neuroquinina B/metabolismo , Preeclampsia/etiología , Receptores de Neuroquinina-3/fisiología , Animales , Biomarcadores , Proteína C-Reactiva/análisis , Proteína C-Reactiva/toxicidad , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Modelos Animales de Enfermedad , Método Doble Ciego , Femenino , Humanos , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fosforilcolina/metabolismo , Placenta/patología , Preeclampsia/inducido químicamente , Preeclampsia/metabolismo , Preeclampsia/patología , Embarazo , Unión Proteica , Procesamiento Proteico-Postraduccional , Quinolinas/farmacología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Receptores de Neuroquinina-3/antagonistas & inhibidores , Receptores de Neuroquinina-3/metabolismo , Proteínas Recombinantes/toxicidad , Método Simple Ciego , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre
8.
J Biol Chem ; 287(46): 38980-91, 2012 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-22988242

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Citidina Trifosfato/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Catálisis , Dominio Catalítico , Citidililtransferasa de Colina-Fosfato/química , Biología Computacional/métodos , Activación Enzimática , Silenciador del Gen , Cinética , Lípidos/química , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , alfa-Sinucleína/química
9.
J Cell Sci ; 124(Pt 24): 4253-66, 2011 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-22223883

RESUMEN

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Membrana Nuclear/ultraestructura , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Animales , Núcleo Celular/ultraestructura , Células Cultivadas , Citidililtransferasa de Colina-Fosfato/análisis , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Lamina Tipo A , Lamina Tipo B/análisis , Proteínas de la Membrana/antagonistas & inhibidores , Metaloendopeptidasas/antagonistas & inhibidores , Ratones , Mitosis , Membrana Nuclear/química , Membrana Nuclear/enzimología , Poro Nuclear/ultraestructura , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Prenilación , Precursores de Proteínas/análisis , Precursores de Proteínas/química
10.
Biochim Biophys Acta ; 1801(11): 1184-94, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20647050

RESUMEN

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.


Asunto(s)
Núcleo Celular/metabolismo , Citidililtransferasa de Colina-Fosfato/fisiología , Enzimas/química , Fosfatidilcolinas/biosíntesis , Animales , Línea Celular , Citidililtransferasa de Colina-Fosfato/química , Citoplasma/metabolismo , Perros , Retículo Endoplásmico/metabolismo , Silenciador del Gen , Lamina Tipo A/química , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Transporte de Proteínas , Factores de Tiempo
11.
Am J Respir Cell Mol Biol ; 43(1): 74-87, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19684306

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCTalpha) plays a key role in the biosynthesis of surfactant phosphatidylcholine. In this study, we investigated the role of its membrane-binding (M) domain in modulating its structure, function, and cellular distribution. Multiple enhanced green fluorescent protein-CCTalpha constructs were generated to evaluate the subcellular distribution in A549 cells. The M domain targeted CCTalpha to the perinuclear (membrane-rich) region. Microinjections with glutathione-S-transferase fusion protein containing the M domain corroborated the perinuclear targeting. Deletion of the M domain or substitutions of the hydrophobic residues with arginine/serine in the VEEKS(267-277) motif of the M domain resulted in a nuclear appearance and indented nuclei. Membrane binding of CCTalpha decreased gradually as the number of positively charged arginine residues increased in the VEEKS motif. To identify whether membrane-protein interactions cause structural alterations in CCTalpha, we visualized the protein in the absence and presence of lipids by transmission electron microscopy. These studies revealed that CCTalpha forms a dimer-like complex that condenses upon binding to lipid vesicles, but not lipid monolayers. The influence of the M domain on CCTalpha activity was assessed in transgenic mice overexpressing the N-terminal catalytic domain (CCTalpha(1-239)), N-terminal catalytic plus M domain (CCTalpha(1-290)), or full-length CCTalpha(1-367) in fetal type II cells by using the surfactant protein C promoter. Only overexpression of CCTalpha(1-367) increased surfactant phosphatidylcholine synthesis. Thus, the M domain influences membrane binding, cellular distribution, and topology of CCTalpha, but the domain alone is not sufficient to confer CCT activity in alveolar type II cells in vivo.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/fisiología , Pulmón/citología , Alveolos Pulmonares/metabolismo , Animales , Dominio Catalítico , Línea Celular Tumoral , Humanos , Lípidos/química , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión/métodos , Mutación , Fosfatidilcolinas/química , Estructura Terciaria de Proteína , Ratas , Relación Estructura-Actividad
12.
Prog Lipid Res ; 47(3): 204-20, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18295604

RESUMEN

Phosphatidylcholine biosynthesis in animal cells is primarily regulated by the rapid translocation of CTP:phosphocholine cytidylyltransferase alpha between a soluble form that is inactive and a membrane-associated form that is activated. Until less than 10 years ago there was no information on the transcriptional regulation of phosphatidylcholine biosynthesis. Research has identified the transcription factors Sp1, Rb, TEF4, Ets-1 and E2F as enhancing the expression of the cytidylyltransferase and Net as a factor that represses cytidylyltransferase expression. Key transcription factors involved in cholesterol or fatty acid metabolism (SREBPs, LXRs, PPARs) do not have a major role in transcriptional regulation of the cytidylyltransferase. Rather than being linked to cholesterol or energy metabolism, regulation of the cytidylyltransferase is linked to the cell cycle, cell growth and differentiation. Transcriptional regulation of phospholipid biosynthesis is more elegantly understood in yeast and involves responses to inositol, choline and zinc in the culture medium.


Asunto(s)
Fosfatidilcolinas/biosíntesis , Transcripción Genética , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/fisiología , Biología Computacional , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosfatidilcolinas/genética , Fosfolípidos/genética , Proteína Proto-Oncogénica c-ets-1/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción Sp1/fisiología
13.
J Biol Chem ; 282(46): 33494-33506, 2007 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-17804406

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCTalpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTalpha protein stability are unknown. An NH(2)-terminal PEST sequence within CCTalpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTalpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTalpha, whereas CaM small interfering RNA accentuated CCTalpha degradation by calpains. CaM bound CCTalpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Gln(243) not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTalpha using a structurally similar molecular signature that profoundly affects CCTalpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under proinflammatory stress.


Asunto(s)
Calmodulina/química , Calmodulina/fisiología , Citidililtransferasa de Colina-Fosfato/fisiología , Adenoviridae/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calpaína/química , Citidililtransferasa de Colina-Fosfato/química , Técnicas de Transferencia de Gen , Glutatión Transferasa/metabolismo , Lipoproteínas/química , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Unión Proteica , Proteínas/química , ARN Interferente Pequeño/metabolismo , Técnicas del Sistema de Dos Híbridos
14.
Mol Cell Biol ; 25(8): 3357-63, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15798219

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (PtdCho). Multiple CCT isoforms, CCTalpha, CCTbeta2, and CCTbeta3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTalpha in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a-/- zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTalpha transcripts are significantly more abundant than transcripts encoding CCTbeta or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTalpha transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTbeta and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTbeta (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTalpha expression in adult liver and other tissues from Pcyt1a+/- heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a+/- mice. Liver PtdCho mass was the same in Pcyt1a+/+ and Pcyt1a+/- adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTalpha expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Genes Letales , Animales , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Embrión de Mamíferos/química , Desarrollo Embrionario/genética , Exones/genética , Femenino , Eliminación de Gen , Marcación de Gen , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/fisiología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Fosfatidilcolinas/análisis , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa , Embarazo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Distribución Tisular , Transcripción Genética
15.
Biochim Biophys Acta ; 1733(1): 53-66, 2005 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-15749057

RESUMEN

Phosphatidylcholine is a prominent constituent of eukaryotic and some prokaryotic membranes. This Perspective focuses on the two enzymes that regulate its biosynthesis, choline kinase and CTP:phosphocholine cytidylyltransferase. These enzymes are discussed with respect to their molecular properties, isoforms, enzymatic activities, and structures, and the possible molecular mechanisms by which they participate in regulation of phosphatidylcholine levels in the cell.


Asunto(s)
Colina Quinasa/fisiología , Citidililtransferasa de Colina-Fosfato/fisiología , Fosfatidilcolinas/biosíntesis , Secuencia de Aminoácidos , Animales , Bacterias/metabolismo , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Colina Quinasa/genética , Citidililtransferasa de Colina-Fosfato/genética , Células Eucariotas/metabolismo , Datos de Secuencia Molecular , Alineación de Secuencia , Homología Estructural de Proteína , Terminología como Asunto
16.
J Biol Chem ; 279(53): 55946-57, 2004 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-15498769

RESUMEN

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCTalpha in lung surfactant maturation, we overexpressed CCTalpha(1-367) using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCTalpha(1-367) increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-(14)C]glucose demonstrated that overexpression of CCTalpha(1-367) in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCTalpha(1-239) or CCTalpha(239-367). Glycogen synthesis and content were increased in fetal type II cells expressing CCTalpha(239-367) but not CCTalpha(1-239)(.) We conclude that overexpression of CCTalpha increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/fisiología , Glucógeno/metabolismo , Fosfatidilcolinas/química , Animales , Western Blotting , Colina/metabolismo , Genotipo , Glucosa/metabolismo , Immunoblotting , Rayos Láser , Pulmón/metabolismo , Pulmón/patología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Microscopía Fluorescente , Modelos Genéticos , Fenotipo , Fosfatidilcolinas/metabolismo , Regiones Promotoras Genéticas , Conformación Proteica , Estructura Terciaria de Proteína , Surfactantes Pulmonares/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transgenes
17.
J Biol Chem ; 279(45): 47402-10, 2004 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-15331603

RESUMEN

CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. Hepatic cells express both an alpha and a beta2 isoform of CT and can also synthesize phosphatidylcholine via the sequential methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase. To ascertain the functional importance of CTalpha, we created a mouse in which the hepatic CTalpha gene was specifically inactivated by the Cre/LoxP procedure. In CTalpha knockout mice, hepatic CT activity (due to residual CTbeta2 activity as well as activity in nonhepatic cells) was 15% of normal, whereas phosphatidylethanolamine N-methyltransferase activity was elevated 2-fold compared with controls. Lipid analyses of the liver indicated that female knockout mice had reduced phosphatidylcholine levels and accumulated triacylglycerols. The plasma phosphatidylcholine concentration was reduced in the CTalpha knockout (independent of gender), as were levels of high density lipoproteins (cholesterol and apoAI) and very low density lipoproteins (triacylglycerols and apoB100). Experiments in which mice were injected with Triton WR1339 indicated that apoB secretion was decreased in hepatic-specific CTalpha knockout mice compared with controls. These results suggest an important role for hepatic CTalpha in regulating both hepatic and systemic lipid and lipoprotein metabolism.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/genética , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Animales , Apolipoproteínas B/metabolismo , Bilis/metabolismo , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Citidililtransferasa de Colina-Fosfato/química , Citidililtransferasa de Colina-Fosfato/fisiología , Detergentes/farmacología , Femenino , Eliminación de Gen , Homocigoto , Immunoblotting , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosfatidilcolinas/química , Polietilenglicoles/farmacología , Isoformas de Proteínas , Factores Sexuales , Factores de Tiempo , Triglicéridos/metabolismo
19.
J Biol Chem ; 275(45): 35368-76, 2000 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-10944538

RESUMEN

Macrophages in atherosclerotic lesions accumulate excess free cholesterol (FC) and phospholipid. Because excess FC is toxic to macrophages, these observations may have relevance to macrophage death and necrosis in atheromata. Previous work by us showed that at early stages of FC loading, when macrophages are still healthy, there is activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), and accumulation of PC mass. We hypothesized that this is an adaptive response, albeit transient, that prevents the FC:PC ratio from reaching a toxic level. To test this hypothesis directly, we created mice with macrophage-targeted disruption of the major CT gene, CTalpha, using the Cre-lox system. Surprisingly, the number of peritoneal macrophages harvested from CTalpha-deficient mice and their overall health under normal culture conditions appeared normal. Moreover, CT activity and PC biosynthesis and in vitro CT activity were decreased by 70-90% but were not absent. As a likely explanation of this residual activity, we showed that CTbeta2, a form of CT that arises from another gene, is induced in CTalpha-deficient macrophages. To test our hypothesis that increased PC biosynthesis is an adaptive response to FC loading, the viability of wild-type versus CTalpha-deficient macrophages under control and FC-loading conditions was compared. After 5 h of FC loading, death increased from 0.7% to only 2.0% in wild-type macrophages but from 0. 9% to 29.5% in CTalpha-deficient macrophages. These data offer the first molecular genetic evidence that activation of CTalpha and induction of PC biosynthesis in FC-loaded macrophages is an adaptive response. Furthermore, the data reveal that CTbeta2 in macrophages is induced in the absence of CTalpha and that a low level of residual CT activity, presumably due to CTbeta2, is enough to keep the cells viable in the peritoneum in vivo and under normal culture conditions.


Asunto(s)
Colesterol/farmacología , Citidililtransferasa de Colina-Fosfato/metabolismo , Citidililtransferasa de Colina-Fosfato/fisiología , Macrófagos/enzimología , Animales , Supervivencia Celular , Citidililtransferasa de Colina-Fosfato/genética , Embrión de Mamíferos/metabolismo , Immunoblotting , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Genéticos , Mutagénesis Insercional , Necrosis , Fosfatidilcolinas/biosíntesis , Isoformas de Proteínas , ARN Mensajero/metabolismo , Recombinación Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Factores de Tiempo
20.
Am J Respir Cell Mol Biol ; 22(1): 116-24, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10615073

RESUMEN

Disaturated phosphatidylcholine (DSPC) is the predominate phospholipid component of lung surfactant. In the alveolar type II cell, the cytidine diphosphocholine (CDP-choline) pathway is the major biosynthetic pathway for DSPC. To investigate the hypothesis that phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the CDP-choline pathway, rat alveolar type II cells or lung tumor-derived cell lines (A549 or H441) with type II cell features were transfected with CT complementary DNA (cDNA). Cell fractions were subsequently assayed for CT protein and activity, and cell rates of DSPC synthesis were determined. In all cases, cell CT protein and activity were increased after transfection with CT cDNA but not after control transfection. Rat type II cells, but not A549 or H441 cells, increased the rate of DSPC synthesis after transfection with CT cDNA. Exposure of type II cells transfected with CT cDNA to palmitic acid resulted in a further increase in CT protein and activity. Exposure to dexamethasone resulted in increased CT protein and activity and increased synthesis of DSPC. The results confirm that CT has a rate-limiting and regulatory role in the synthesis of type II cell DSPC, and raise possibilities for novel therapeutic interventions.


Asunto(s)
Citidililtransferasa de Colina-Fosfato/biosíntesis , Fosfatidilcolinas/biosíntesis , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/metabolismo , Animales , Fraccionamiento Celular , Línea Celular , Citidililtransferasa de Colina-Fosfato/fisiología , Citosol/enzimología , Dexametasona/farmacología , Epitelio/efectos de los fármacos , Epitelio/enzimología , Epitelio/metabolismo , Immunoblotting , Plásmidos/genética , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Ratas , Transfección
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...